Methods of treating neuropsychiatric disorders

ABSTRACT

The present disclosure is directed to a method of treating a neuropsychiatric disorder. This method involves selecting a subject having the neuropsychiatric disorder and administering to the selected subject a preparation of glial progenitor cells at a dosage effective to treat the neuropsychiatric disorder in the subject. Another aspect of the disclosure is directed to a method of treating a neuropsychiatric disorder that includes selecting a subject having the neuropsychiatric disorder and administering, to the selected subject, a potassium (K + ) channel activator at a dosage effective to restore normal brain interstitial glial K +  levels in the selected subject and treat the neuropsychiatric disorder is also disclosed.

This application is a divisional of U.S. Pat. Application Serial No.16/612,529, filed Nov. 11, 2019, which is a national stage applicationunder 35 U.S.C. § 371 of PCT Application No. PCT/US2018/031961, filedMay 10, 2018, which claims the benefit of U.S. Provisional Pat.Application Serial No. 62/504,340, filed May 10, 2017, which is herebyincorporated by reference in its entirety.

This invention was made with government support under MH099578 andMH104701 awarded by National Institutes of Health. The government hascertain rights in the invention.

FIELD

The present application relates to methods of treating neuropsychiatricdisorders.

BACKGROUND

There are a number of uniquely human neurological disorders, whosephylogenetic appearance parallels that of human glial evolution, whichaccelerated with the appearance of hominids (Oberheim et al.,“Astrocytic Complexity Distinguishes the Human Brain,” Trends inNeurosciences 29:1-10 (2006); Oberheim et al., “Uniquely HominidFeatures of Adult Human Astrocytes,” The Journal of Neuroscience : TheOfficial Journal of the Society for Neuroscience 29:3276-3287 (2009);Horrobin, D. F., “Schizophrenia: The Illness That Made Us Human,” MedHypotheses 50:269-288 (1998)). In particular, astroglial complexity andpleomorphism increased significantly with hominid evolution, whichsuggests an association between human glial evolution and thedevelopment of human-selective neurological disorders. Indeed, a numberof both genome-wide association and differential expression studies havehighlighted the frequent dysregulation of glial-selective genes, bothastrocytic and oligodendrocytic, in, for example, schizophrenia (Walshet al., “Rare Structural Variants Disrupt Multiple Genes inNeurodevelopmental Pathways in Schizophrenia,” Science 320:539-543(2008); Aberg et al., “Human QKI, A Potential Regulator of mRNAExpression of Human Oligodendrocyte-Related Genes Involved inSchizophrenia,” Proceedings of the National Academy of Sciences of theUnited States of America 103:7482-7487 (2006); Roy et al., “Loss of erbBSignaling in Oligodendrocytes Alters Myelin and Dopaminergic Function, APotential Mechanism for Neuropsychiatric Disorders,” Proceedings of theNational Academy of Sciences of the United States of America104:8131-8136 (2007); Takahashi et al., “Linking Oligodendrocyte andMyelin Dysfunction to Neurocircuitry Abnormalities in Schizophrenia,”Prog Neurobiol 93:13-24 (2011); Georgieva et al., “Convergent EvidenceThat Oligodendrocyte Lineage Transcription Factor 2 (OLIG2) andInteracting Genes Influence Susceptibility to Schizophrenia,”Proceedings of the National Academy of Sciences of the United States ofAmerica 103:12469-12474 (2006); Hof et al., “Molecular and CellularEvidence for an Oligodendrocyte Abnormality in Schizophrenia,” NeurochemRes 27:1193-1200 (2002); Hakak et al., “Genome-Wide Expression AnalysisReveals Dysregulation of Myelination-Related Genes in ChronicSchizophrenia,” Proceedings of the National Academy of Sciences of theUnited States of America 98:4746-4751 (2001)).

Patients with schizophrenia are typically characterized by a relativepaucity of white matter and often frank hypomyelination (Takahashi etal., “Linking Oligodendrocyte and Myelin Dysfunction to NeurocircuitryAbnormalities in Schizophrenia,” Prog Neurobiol 93:13-24 (2011); Connoret al., “White Matter Neuron Alterations in Schizophrenia and RelatedDisorders,” International Journal of Developmental Neuroscience : TheOfficial Journal of the International Society for DevelopmentalNeuroscience 29:325-334 (2011); McIntosh et al., “White MatterTractography in Bipolar Disorder and Schizophrenia,” BiologicalPsychiatry 64:1088-1092 (2008); Maniega et al., “A Diffusion Tensor MRIStudy of White Matter Integrity in Subjects at High Genetic Risk ofSchizophrenia,” Schizophrenia Research 106:132-139 (2008); Fields, R.D., White Matter in Learning, Cognition and Psychiatric Disorders,“Trends in Neurosciences 31:361-370 (2008); Gogtay et al.,“Three-Dimensional Brain Growth Abnormalities in Childhood-OnsetSchizophrenia Visualized by Using Tensor-Based Morphometry,” Proceedingsof the National Academy of Sciences of the United States of America105:15979-15984 (2008)). A number of both pathological and neuroimagingstudies have highlighted deficiencies in both oligodendroglial densityand myelin structure in affected patients (Fields, R. D., White Matterin Learning, Cognition and Psychiatric Disorders, “Trends inNeurosciences 31:361-370 (2008); Xia et al., “Behavioral Sequelae ofAstrocyte Dysfunction: Focus on Animal Models of Schizophrenia,”Schizophrenia Research (2014); Rapoport et al., “The NeurodevelopmentalModel of Schizophrenia: Update 2005,” Molecular Psychiatry 10:434-449(2005); Langmead et al., “Fast Gapped-Read Alignment with Bowtie 2,”Nature Methods 9:357-359 (2012)), including at the ultrastructural level(Uranova et al., “Ultrastructural Alterations of Myelinated Fibers andOligodendrocytes in the Prefrontal Cortex in Schizophrenia: A PostmortemMorphometric Study,” Schizophrenia Research and Treatment 2011:325789(2011); Uranova et al., “The Role of Oligodendrocyte Pathology inSchizophrenia,” Int J Neuropsychopharmacol 10:537-545 (2007); Pruitt etal., “NCBI Reference Sequences (RefSeq): A Curated Non-RedundantSequence Database of Genomes, Transcripts and Proteins,” Nucleic AcidsResearch 35:D61-65 (2007)). Furthermore, recent studies have emphasizedthe role of oligodendrocytes in the metabolic support of neurons,suggesting myelin-independent mechanisms whereby oligodendrocyticdysfunction might yield neuronal pathology (Lee et al., “OligodendrogliaMetabolically Support Axons and Contribute to Neurodegeneration,” Nature487:443-448 (2012); Simons et al., “Oligodendrocytes: Myelination andAxonal Support,” Cold Spring Harb Perspect Biol. (2015)). Yet despitegenetic, cellular, pathological, and radiological studies that havecorrelated glial and myelin pathology with schizophrenia, the prevailingview is that that clinical hypomyelination among schizophrenics issecondary to neuronal pathology. Thus, the contribution ofcell-autonomous glial dysfunction to schizophrenia has not been wellstudied, and consequently therapies targeting such dysfunctions have yetto be proposed.

The present disclosure is directed to overcoming these and otherdeficiencies in the art.

SUMMARY

One aspect of the present disclosure relates to a method of treating aneuropsychiatric disorder. This method involves selecting a subjecthaving the neuropsychiatric disorder and administering to the selectedsubject a preparation of glial progenitor cells at a dosage effective totreat the neuropsychiatric disorder in the subject.

Another aspect of the present disclosure relates to a method of treatinga neuropsychiatric disorder. This method includes selecting a subjecthaving the neuropsychiatric disorder and administering, to the selectedsubject, a K⁺ channel activator at a dosage effective to restore normalbrain interstitial glial K⁺ levels in the selected subject and treat theneuropsychiatric disorder.

Another aspect of the present disclosure relates to a non-human animalmodel of a neuropsychiatric disorder. This non-human mammal has at least30% of all of its glial cells in its corpus callosum being human glialcells derived from a human patient with a neuropsychiatric disorderand/or at least 5% of all of its glial cells in the white matter of itsbrain and/or brain stem being human glial cells derived from a humanpatient with a neuropsychiatric disorder.

Applicants have established that the contribution of cell autonomousglial dysfunction neurological disease can be investigated using a novelmodel of human glial chimeric mice (Windrem et al., “NeonatalChimerization with Human Glial Progenitor Cells Can Both Remyelinate andRescue the Otherwise Lethally Hypomyelinated Shiverer Mouse,” Cell StemCell 2:553-565 (2008); Han et al., “Forebrain Engraftment by Human GlialProgenitor Cells Enhances Synaptic Plasticity and Learning in AdultMice,” Cell Stem Cell 12:342-353 (2013); Goldman et al., “ModelingCognition and Disease Using Human Glial Chimeric Mice,” Glia63:1483-1493 (2015), which are hereby incorporated by reference in theirentirety) paired with the development of protocols for generatingbipotential astrocyte-oligodendrocyte glial progenitor cells (GPCs) frompatient-specific human induced pluripotent stem cells (hiPSCs) (Wang etal., “Human iPSC-Derived Oligodendrocyte Progenitor Cells Can Myelinateand Rescue a Mouse Model of Congenital Hypomyelination,” Cell Stem Cell12:252-264 (2013), which is hereby incorporated by reference in itsentirety). In these human glial chimeric mouse brains, the majority ofresident glia are replaced by human glia and their progenitors (Windremet al., “A Competitive Advantage by Neonatally Engrafted Human GlialProgenitors Yields Mice Whose Brains are Chimeric for Human Glia,” TheJournal of Neuroscience: The Official Journal of the Society forNeuroscience 34:16153-16161 (2014), which is hereby incorporated byreference in its entirety), allowing human glial physiology, geneexpression, and effects on neurophysiological function to be assessed invivo, in live adult mice (Han et al., “Forebrain Engraftment by HumanGlial Progenitor Cells Enhances Synaptic Plasticity and Learning inAdult Mice,” Cell Stem Cell 12:342-353 (2013), which is herebyincorporated by reference in its entirety). As described herein, theglial chimeric model was used to assess the contribution of human gliato schizophrenic disease phenotype. To this end, hGPCs were preparedfrom iPSCs derived from fibroblasts taken from either juvenile-onsetschizophrenic (SCZ) patients or their normal controls. Differential geneexpression of SCZ hGPCs was assessed relative to those of normalsubjects, and the cells were transplanted into immunodeficient neonatalmice to produce patient-specific human glial chimeric mice. The glialchimeric mice were then analyzed in regard to the effects of SCZderivation on astrocytic and oligodendrocytic differentiations in vivo,as well as on behavioral phenotype, and the data thereby obtainedcorrelated to disease-associated gene expression. Using this model ofhuman specific neuropsychiatric disease, applicants have identifiednovel therapeutic approaches for the treatment of human neuropsychiatricdisorders and conditions that are described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the functional and genomic assessment ofschizophrenia-derived glial progenitor cells. This schematic summarizesthe steps involved in the analysis of glial progenitor cells derivedfrom individuals with juvenile-onset schizophrenia, compared to GPCsderived from behaviorally-normal controls. The major output data includeeffects of SCZ origin on in vivo oligodendrocyte maturation andmyelination (FIG. 3 ); in vivo astrocyte differentiation and phenotype(FIG. 5 ); in vitro differential gene expression (FIG. 6 ); andbehavioral phenotype of the human glial chimeric host animals (FIG. 10).

FIGS. 2A-2H show CD140a+ glial progenitor cells are efficiently producedfrom both SCZ and normal hiPSCs. Flow cytometry for CD140a/PDGFαR+ glialprogenitor cells (right plots, compared to unstained gating controls onleft), reveals dominant proportions of CD140a-defined cells in bothnormal control patient-derived (top, FIGS. 2A-2B) and SCZ-derived(bottom, FIGS. 2C-2D) preparations. FIGS. 2A-2C and 2B-2D were run asmatched pairs; FIGS. 2A and 2C show 177 and 168 days in vitro (DIV);FIGS. 2B and 2D show188 and 196 DIV. FIGS. 2E-2H show a representativepost-FACS preparation of CD140a-sorted cells. FIG. 2E is a phase imageof cells immunostained for both olig2 (FIG. 2F, red) and PDGFRα (FIGS.2G; 2H, merged). These plots were typical of GPC cultures of both normaland SCZ-derived hiPSC lines. The sorted populations were used forgenomics assessment, while both sorted and unsorted cells were used fortransplantation, with no evident performance differences between thetwo.

FIGS. 3A-3J show schizophrenia-derived hGPCs exhibit aberrant dispersaland relative hypomyelination. Human iPSC GPC chimeras were establishedby neonatal hGPC injection into shiverer mice. Chimeric mice weresacrificed at 19 weeks. GPCs derived from a control subject (FIG. 3A)dispersed primarily in the major white matter tracts, whereasSCZ-derived GPCs (15 year old male) (FIG. 3B) showed less white matterresidence and more rapid cortical infiltration. FIGS. 3C-3D are sagittalsections that reveal callosal myelination by SCZ GPCs (FIG. 3D) was lessdense than that by control hGPCs (FIG. 3C). FIGS. 3E-3F show higherpower images from chimeric mice engrafted with hGPCs from 4 controlpatients (FIG. 3E) vs. chimeric mice engrafted with hGPCs from 4different SCZ patients (FIG. 3F). FIG. 3G shows MBP luminance confirmedthe greater callosal myelination of CTRL GPC-engrafted vs. SCZGPC-engrafted mice at 19 weeks (means of 4 different SCZ and CTRLpatients each, n>3 mice/patient) (p=0.0002, t-test). FIG. 3H shows thatabsolute donor cell densities were lower in SCZ than controlhGPC-engrafted corpus callosum (p<0.0001, t-test), as were the densitiesof olig2⁺ hGPCs and oligodendroglia (FIG. 3I) (p=0.0064, t-test) andtransferrin (TFN)⁺ oligodendroglia (FIG. 3J) (p<0.0001, t-test).

FIGS. 4A-4B show schizophrenia-derived GPCs exhibit aberrant dispersalin vivo. The dispersal patterns of GPCs produced from SCZ patientstypically differed from that of iPSC hGPCs derived from normal patients,in that SCZ GPCs did not remain and expand within the white matterbefore progressing to cortical infiltration, as was otherwise invariablythe case with normal GPCs. FIGS. 4A-4B show 4 mice each implanted witheither control subject-derived (line 22) or SCZ patient-derived (line51) hGPCs. All SCZ hGPC-engrafted mice show disproportionate hGPC entryinto the cortical and striatal gray matter, with less expansion andhence less net engraftment in the forebrain white matter tracts. Thisdifference in hGPC dispersal pattern was noted consistently in all 4 SCZlines assessed in vivo, each derived from a different patient, relativeto their matched 4 control lines, similarly obtained from distinctpatients (see FIG. 3 ).

FIGS. 5A-5J show astrocytic differentiation is impaired in schizophreniahGPC chimeric brain. Human iPSC GPC chimeras were established inimmunodeficient shiverer hosts and sacrificed at 19 weeks, forastrocytic differentiation assessment. FIGS. 5A-5B are representativeimages of the corpus callosum of mice neonatally injected with iPSC GPCsderived from either control (FIG. 5A, line 22) or schizophrenic (FIG.5B, line 164) subjects (human nuclear antigen, green; glial fibrillaryacidic protein, red). FIG. 5A shows control hiPSC GPCs from all testedpatients rapidly differentiated as GFAP+ astrocytes with dense fiberarrays in both callosal white and cortical gray matter. FIG. 5B, incontrast, shows SCZ GPCs were slow to mature, with delayed GFAPexpression. At 19 weeks, GFAP+ astrocyte densities were significantlygreater in mice chimerized with control than SCZ-derived GPCs, both as agroup (FIG. 5C), and when analyzed line-by-line (FIG. 5D). This was notjust a function of less callosal engraftment, as the proportion of humandonor cells that developed GFAP and astrocytic phenotype wassignificantly lower in SCZ- than control GPC-engrafted mice (FIG. 5E).Sholl analysis of individual astroglial morphologies (Sholl, D.A.,“Dendritic Organization in the Neurons of the Visual and Motor Corticesof the Cat,” J. Anat. 87:387-406 (1953), which is hereby incorporated byreference in its entirety), as imaged in 150 µm sections andreconstructed in 3D by Neurolucida (FIG. 5J), revealed that astrocytesin SCZ hGPC chimeras differed significantly from their controlhGPC-derived counterparts, with fewer primary processes (FIG. 5F), lessproximal branching (FIG. 5G), and longer distal fibers (FIG. 5H). Whenthe 3-D tracings (FIG. 5J) were assessed by Fan-in radial analysis (MBFBiosciences) (Dang et al., “Formoterol, a Long-acting Beta2 AdrenergicAgonist, Improves Cognitive Function and Promotes Dendritic Complexityin a Mouse Model of Down Syndrome,” Biol. Psychiatry 75:179-188 (2014),which is hereby incorporated by reference in its entirety), controlastrocytic processes were noted to extend uniformly in all directions,but SCZ astrocyte processes left empty spaces, indicative of adiscontiguous domain structure (FIG. 5I). ***p<0.0001, by t-test (FIGS.5C, 5E, 5F, 5H; by 2-way ANOVA in FIG. 5D; **p<0.002 in FIG. 5I;p<0.0001 by non-linear comparison in FIG. 5G. Scale, FIGS. 5A-5B = 50µm, FIG. 5J = 25 µm.

FIGS. 6A-6G show schizophrenia-derived hGPCs suppress glialdifferentiation-associated gene expression. RNA sequence analysisreveals differential gene expression by SCZ hGPCs. FIG. 6A shows anintersection of lists of differentially expressed genes (DEGs)(log2-fold change >1.00, FDR 5%) obtained by comparison of hGPCs derivedfrom 4 different schizophrenia patients, compared to pooled controlhGPCs. FIG. 6B is a network representation of functional annotations forthe intersection gene list shown in FIG. 6A. In the upper network, greenand red nodes represent down- and up-regulated genes, respectively, andwhite nodes represent significantly associated annotation terms(FDR-corrected p< 0.01; annotation terms include GO:BP, GO:MF, pathways,and gene families, and nodes are sized by degree). Lower networkhighlights 4 highly interconnected modules identified by communitydetection. FIG. 6C shows top annotation terms identified for each modulein FIG. 6B. FIG. 6D is a heatmap representation of 12 conserveddifferentially expressed genes that are associated to module 1 (grey inFIG. 6B, 32.4%), which includes annotations related to neurotransmitterreceptor and gated channel activity. FIG. 6E is a heatmap representationof 15 conserved differentially expressed genes that are associated tomodule 2 (orange in FIG. 6B, 28.7%), which comprises annotations relatedto cell-to-cell signaling and synaptic transmission. FIG. 6F is aheatmap representation of 21 conserved differentially expressed genesthat are associated to module 3 (dark blue in FIG. 6B, 28.7%);annotations related to CNS and glial differentiation and development.FIG. 6G is a heatmap representation of 4 conserved differentiallyexpressed genes that are associated to module 4 (light blue in FIG. 6B,10.2%), with annotations related to myelination and lipid biosynthesis.The absolute expression in heatmaps is shown in UQ-normalized,log2-transformed counts (Li et al., “Comparing the Normalization Methodsfor the Differential Analysis of Illumina High-Throughput RNA-Seq Data,”BMC Bioinformatics 16:347 (2015), which is hereby incorporated byreference in its entirety).

FIGS. 7A-7R show heat maps of significantly dysregulated genes inschizophrenic relative to control hiPSC GPCs. Expression patterns forshared genes differentially expressed by hiPSC GPCs derived from 4schizophrenic patients, relative to the pooled gene expression patternof hGPCs derived from 3 control-derived iPSCs (log2 fold change >1.0,FDR 5%, 118 genes total) are shown. The dysregulated genes were manuallyannotated and grouped into relevant sets based on their function andcellular localization. Each heat map visualizes UQ-normalized,log2-transformed counts of genes grouped into the following functionalcategories, comprising genes encoding: (FIG. 7A) transcriptionregulators, zinc finger proteins, and other nucleus-associated proteins;(FIG. 7B) glial differentiation-associated proteins; (FIG. 7C)myelin-related genes and transcription factors; (FIG. 7D) Wnt pathwayeffectors; (FIG. 7E) metabolic enzymes; (FIG. 7F) lipid and lipoproteinmetabolism; (FIG. 7G) kinases and phosphatases; (FIG. 7H) adhesionmolecules, cadherins, and astrotactins; (FIG. 7I) GPCR signalintermediates; (FIG. 7J) growth factors; (FIG. 7K) cytokines; (FIG. 7L)cell signaling and synaptic proteins; (FIG. 7M) ion channels; (FIG. 7N)transporters; (FIG. 7O) extracellular matrix constituents; (FIG. 7P)other transmembrane proteins; (FIG. 7Q) other cytoplasmic andmembrane-bound proteins; and (FIG. 7R) unannotated genes, open readingframes, and long intergenic non-coding RNAs.

FIG. 8 shows expression of selected genes dysregulated in SCZ-derivedGPCs as identified by RNA-seq analysis assessed by TaqMan Low DensityArray (TLDA) RT-qPCR and compared against control GPCs. Expression datawere normalized to GAPDH endogenous control. Mean ddCt values andstandard error ranges calculated from 4 pooled SCZ GPC lines (n = 19)against 3 pooled control GPC lines (n = 10) are shown. The difference ofexpression in SCZ and control GPCs was assessed by paired t-testfollowed by multiple testing correction by Benjamini-Hochberg (BH)procedure (*** = p <0.01, ** = p < 0.05, * = p <0.1). 48 genes wereassessed. 45 genes are shown, excluding the endogenous control and genesthat had high proportion of undetermined and unreliable reactions, LRFN1and NEUROD6. The vast majority of genes were confirmed as dysregulatedin SCZ-derived GPCs. Analysis of TLDA data was performed inExpressionSuite Software version 1.1 supplied by Applied Biosciences.

FIG. 9 shows neurexin-1 expression was suppressed in SCZ hGPCs. Westernblots revealed that neurexin-1 protein was abundantly expressed by humanGPCs purified by CD140a-directed FACS, and that neurexin-1 levels werelower in otherwise matched SCZ hGPCs (line 51 SCZ hGPCs vs. line 22 CTRLhGPCs).

FIGS. 10A-10G show schizophrenia-derived human glial chimeras havesignificant behavioral abnormalities. FIGS. 10A-10E show behavioraltests that were performed in mice chimerized with one of 3 SCZ or 3control hGPC lines, each line from a different patient. 7-20 recipientmice were tested per cell line, males and females equally. FIG. 10Ashows prepulse inhibition studies. Normally-myelinated rag1-/- miceengrafted with SCZ hGPCs had reduced auditory pre-pulse inhibition (PPI)at all volumes of pre-pulse (FIG. 10A). The extent of PPI differedsignificantly between control (n=13) and SCZ (n=27) hGPC-engraftedanimals (p=0.0008 by ANOVA, F=11.76 [1,114]). FIG. 10B shows elevatedplus maze studies. The left panel shows representative heat maps of thecumulated movement of a mouse engrafted with SCZ hGPCs, relative to itsmatched normal hGPC-engrafted control, in the elevated plus maze, a testdesigned to assess anxiety, in which preference for enclosed space andavoidance of open height suggests greater anxiety. The right panel showsmice engrafted with hGPCs from 3 SCZ patients (12 implanted mice each,for n=36 mice total) spent more time in the closed maze arms than didcontrol-engrafted mice (n=36, also derived from 3 patients) (p=0.036,2-tailed t test). FIG. 10C shows sucrose preference studies. SCZGPC-engrafted mice were less likely to prefer sweetened water,suggesting relative anhedonia (p=0.02, Mann-Whitney t-test; n=30 micederived from 3 SCZ lines; n=17 mice from 3 control lines). FIG. 10Dshows 3-chamber socialization test studies. Mice engrafted with hGPCswere placed into the middle chamber of a box divided into 3compartments, one holding an empty cage (bottom, “X” in FIG. 10D) andone containing an unfamiliar mouse (top, filled white circle), thenvideo-tracked for 10 minutes. Mice engrafted with SCZ hGPCs (rightheat-map) avoided strangers more than controls (left heat-map) (p=0.02;3 SCZ lines, 34 mice; 3 control lines, 36 mice). FIG. 10E shows novelobject recognition studies. Mice engrafted with SCZ hGPCs showedsignificantly poorer novel object recognition (p=0.0006; 3 SCZ lines, 19mice; 3 control lines, 28 mice). FIGS. 10F-10G demonstrate the diurnalactivity and sleep patterns of adult mice (70-80 weeks old) engraftedneonatally with either SCZ or CTRL hGPCs were assessed for 72 hrs inclosed chambers (Noldus Ethovision), under continuous video recording.FIG. 10F shows the average distance traveled in meters/hr over a 72 hrperiod calculated and compared between CTRL mice (gray fill, n=8 mice;lines 22 and 17) and SCZ mice (purple fill; n=10, line 52). Time of dayis shown as a 24-hour cycle, with the dark phase indicated by graybackground shading. The SCZ mice were significantly more activethroughout the observation period than CTRL-engrafted mice (p<0.0001,ANOVA, F=19.32 [1,851]. FIG. 10G shows, on the left, sample heat-maps ofone hour of activity during the light phase (16:00 hrs, 2nd day in box),the normal period of sleep for mice. The control mouse (left) remainsinactive for the entire hour, while the SCZ mouse moves about the cageduring much of the hour. As shown on the right, the SCZ mice exhibitedsleep patterns that were fragmented into bouts of shorter duration thantheir normal hGPC- chimeric controls (p=0.0026 by ANOVA, F=12.08 [1,24].Means ± SEM; unpaired, two-tailed Welch-corrected t-tests.

DETAILED DESCRIPTION

One aspect of the present disclosure relates to a method of treating aneuropsychiatric disorder. This method involves selecting a subjecthaving the neuropsychiatric disorder and administering to the selectedsubject a preparation of glial progenitor cells at a dosage effective totreat the neuropsychiatric disorder in the subject.

A “neuropsychiatric disorder” as referred to herein, includes any braindisorder with psychiatric symptoms including, but not limited to,dementia, amnesic syndrome, and personality-behavioral changes.Exemplary neuropsychiatric disorders to be treated using the methodsdescribed herein include, without limitation, schizophrenia, autismspectrum disorders, and bipolar disorder.

Schizophrenia is a chronic and severe mental disorder that affects how aperson thinks, feels, and behaves. To date, there have been severalsuggested staging models of the disorder (Agius et al., “The StagingModel in Schizophrenia, and its Clinical Implications,” Psychiatr.Danub. 22(2):211-220 (2010); McGorry et al., “Clinical Staging: aHeuristic Model and Practical Strategy for New Research and BetterHealth and Social Outcomes for Psychotic and Related Disorders,” Can. J.Psychiatry 55(8):486-497 (2010); Fava and Kellner, “Staging: a NeglectedDimension in Psychiatric Classification,” Acta Psychiatr. Scand.87:225-230 (1993), which are hereby incorporated by reference in theirentirety). However, generally, schizophrenia develops in at least threestages: the prodromal phase, the first episode, and the chronic phase.There is also heterogeneity of individuals at all stages of thedisorder, with some individuals considered ultra-high risk,clinical-high risk, or at-risk for the onset of psychosis (Fusar-Poli etal., “The Psychosis High-Risk State: a Comprehensive State-of-the-ArtReview,” JAMA Psychiatry 70:107-120 (2013), which is hereby incorporatedby reference in its entirety).

The methods described herein are suitable for treating a subject in anystage of schizophrenia, and at any risk level of psychosis. For example,in one embodiment, a subject treated in accordance with the methodsdescribed herein is a subject that is at risk for developingschizophrenia. Such a subject may have one or more genetic mutations inone or more genes selected from ABCA13, ATK1, C4A, COMT, DGCR2, DGCR8,DRD2, MIR137, NOS1AP, NRXN1, OLIG2, RTN4R, SYN2, TOP3B YWHAE, ZDHHC8, orchromosome 22 (22q11) that have been associated with the development ofschizophrenia and may or may not be exhibiting any symptoms of thedisease. In another embodiment, the subject may be in the prodromalphase of the disease and exhibiting one or more early symptoms ofschizophrenia, such as anxiety, depression, sleep disorders, and/orbrief intermittent psychotic syndrome. In another embodiment, thesubject being treated in accordance with the methods described herein isexperiencing psychotic symptoms, e.g., hallucinations, paranoiddelusions, of schizophrenia.

As referred to herein, “Autism Spectrum Disorder” encompasses a group ofconditions including Autistic disorder, Asperger’s disorder, PervasiveDevelopmental Disorder-Not Otherwise Specified, Childhood DisintegrativeDisorder, and Rett’s Disorder, which vary in the severity of symptomsincluding difficulties in social interaction, communication, and unusualbehaviors (McPartland et al., “Autism and Related Disorders,” Handb ClinNeurol 106:407-418 (2012), which is hereby incorporated by reference inits entirety). The methods described herein are suitable for thetreatment of each one of these conditions included in the autismspectrum.

As referred to herein “bipolar disorder” is a group of conditionscharacterized by chronic instability of mood, circadian rhythmdisturbances, and fluctuations in energy level, emotion, sleep, andviews of self and others. “Bipolar disorders” encompasses bipolardisorder type I, bipolar disorder type II, cyclothymic disorder, andbipolar disorder not otherwise specified. Individuals at greatest riskfor developing a bipolar disorder are those with a family history of thecondition. To date, there have been several suggested staging models ofthe disorders (McGorry et al., “Clinical Staging: a Heuristic Model andPractical Strategy for New Research and Better Health and SocialOutcomes for Psychotic and Related Disorders,” Can. J. Psychiatry55(8):486-497 (2010); McNamara et al., “Preventative Strategies forEarly-Onset Bipolar Disorder: Towards a Clinical Staging Model,” CNSDrugs 24:983-996 (2010); Kapczinski et al., “Clinical Implications of aStaging Model for Bipolar Disorders,” Expert Rev Neurother 9:957-966(2009), which are hereby incorporated by reference in their entirety).However, generally, bipolar disorders are progressive conditions whichdevelop in at least three stages: the prodromal phase, the symptomaticphase, and the residual phase.

The methods described herein are suitable for treating subjects havingany of the aforementioned bipolar disorders and subjects in any stage ofa particular bipolar disorder. The methods described herein are suitablefor treating subjects having any of the aforementioned bipolar disordersand subjects in any stage of a particular bipolar disorder. For example,in one embodiment, the subject treated in accordance with the methodsdescribed herein is a subject at the early prodromal phase exhibitingsymptoms of mood lability/swings, depression, racing thoughts, anger,irritability, physical agitation, and anxiety. In another embodiment,the subject treated in accordance with the methods described herein is asubject at the symptomatic phase or the residual phase.

As used herein, the term “subject” expressly includes human andnon-human mammalian subjects. The term “non-human mammal” as used hereinextends to, but is not restricted to, household pets and domesticatedanimals. Non-limiting examples of such animals include primates, cattle,sheep, ferrets, mice, rats, swine, camels, horses, poultry, fish,rabbits, goats, dogs and cats.

In accordance with aspects illustrated herein, the preparation of glialprogenitor cells to be administered to the selected subject may be humanor non-human. In one embodiment, the preparation of glial progenitorcells is a preparation of human glial progenitor cells.

Preferably the glial progenitor cells are bi-potential glial progenitorcells. In one embodiment, the glial progenitor cells are biased toproducing oligodendrocytes. In another embodiment, the glial progenitorcells are biased to producing astrocytes. Methods and markers forproducing and distinguishing astrocyte-biased and oligodendrocyte-biasedglial progenitor cells are described herein.

Glial progenitor cells suitable for use in the methods described herecan be derived from multipotent (e.g., neural stem cells) or pluripotentcells (e.g., embryonic stem cells or induced pluripotent stem cells)using methods known in the art or described herein.

In one embodiment, glial progenitor cells are derived from embryonicstem cells. Embryonic stem cells are derived from totipotent cells ofthe early mammalian embryo and are capable of unlimited,undifferentiated proliferation in vitro. As used herein, the term“embryonic stem cells” refer to a cells isolated from an embryo,placenta, or umbilical cord, or an immortalized version of such a cells,i.e., an embryonic stem cell line. Suitable embryonic stem cell linesinclude, without limitation, lines WA-01 (H1), WA-07, WA-09 (H9), WA-13,and WA-14 (H14) (Thomson et al., “Embryonic Stem Cell Lines Derived fromHuman Blastocytes,” Science 282 (5391): 1145-47 (1998) and U.S. Pat. No.7,029,913 to Thomson et al., which are hereby incorporated by referencein their entirety). Other suitable embryonic stem cell lines includesthe HAD-C100 cell line (Tannenbaum et al., “Derivation of Xeno-free andGMP-grade Human Embryonic Stem Cells - Platforms for Future ClinicalApplications,” PLoS One 7(6):e35325 (2012), which is hereby incorporatedby reference in its entirety, the WIBR4, WIBR5, WIBR6 cel lines (Lengneret al., “Derivation of Pre-x Inactivation Human Embryonic Stem Cell Linein Physiological Oxygen Conditions,” Cell 141(5):872-83 (2010), which ishereby incorporated by reference in its entirety), and the humanembryonic stem cell lines (HUES) lines 1-17 (Cowan et al., “Derivationof Embryonic Stem-Cell Lines from Human Blastocytes,” N. Engl. J. Med.350:1353-56 (2004), which is hereby incorporated by reference in itsentirety).

In one embodiment, glial progenitor cells are derived from inducedpluripotential cells (iPSCs). “Induced pluripotent stem cells” as usedherein refers to pluripotent cells that are derived from non-pluripotentcells, such as somatic cells or tissue stem cells. For example, andwithout limitation, iPSCs can be derived from embryonic, fetal, newborn,and adult tissue, from peripheral blood, umbilical cord blood, and bonemarrow (see e.g., Cai et al., “Generation of Human Induced PluripotentStem Cells from Umbilical Cord Matrix and Amniotic Membrane MesenchymalCells,” J. Biol. Chem. 285(15): 112227-11234 (2110); Giorgetti et al.,“Generation of Induced Pluripotent Stem Cells from Human Cord BloodCells with only Two Factors: Oct4 and Sox2,” Nature Protocols,5(4):811-820 (2010); Streckfuss-Bomeke et al., “Comparative Study ofHuman-Induced Pluripotent Stem Cells Derived from Bone Marrow Cells,Hair Keratinocytes, and Skin Fibroblasts,” Eur. Heart J. doi:10.1093/eurheartj/ehs203 (Jul. 12, 2012); Hu et al., “EfficientGeneration of Transgene-Free Induced Pluripotent Stem Cells from Normaland Neoplastic Bone Marrow and Cord Blood Mononuclear Cells,” Blood doi:10.1182/blood-2010-07-298331 (Feb. 4, 2011); Sommer et al., “Generationof Human Induced Pluripotent Stem Cells from Peripheral Blood using theSTEMCCA Lentiviral Vector,” J. Vis. Exp. 68: e4327 doi: 10.3791/4327(2012), which are hereby incorporated by reference in their entirety).Exemplary somatic cells that can be used include fibroblasts, such asdermal fibroblasts obtained by a skin sample or biopsy, synoviocytesfrom synovial tissue, keratinocytes, mature B cells, mature T cells,pancreatic β cells, melanocytes, hepatocytes, foreskin cells, cheekcells, or lung fibroblasts (see e.g., Streckfuss-Bomeke et al.,“Comparative Study of Human-Induced Pluripotent Stem Cells Derived fromBone Marrow Cells, Hair Keratinocytes, and Skin Fibroblasts,” Eur. HeartJ. doi: 10.1093/eurheartj/ehs203 (2012), which is hereby incorporated byreference in its entirety). Although skin and cheek provide a readilyavailable and easily attainable source of appropriate cells, virtuallyany cell can be used. Exemplary stem or progenitor cells that aresuitable for iPSC production include, without limitation, myeloidprogenitors, hematopoietic stem cells, adipose-derived stem cells,neural stem cells, and liver progenitor cells.

Autologous, allogenic, or xenogenic non-pluripotent cells can be used into produce the iPSCs used to generate the therapeutic glial progenitorcells. Allogenic cells for production of iPSCs, for example, areharvested from healthy donors (i.e., donors not having aneuropsychiatric disorder) and/or donor sources having suitableimmunohistocompatibility. Xenogeneic cells can be harvested from a pig,monkey, or any other suitable mammal for the production if iPSCs.Autologous non-pluripotent cells can also be harvested from the samesubject to be treated. However, such autologous cells require geneticmanipulation and/or other treatment prior to therapeutic administration.In particular, as described herein expression of a number of genes (seeTable 2) are dysregulated in neuropsychiatric disorders. Accordingly,autologous cells are preferably genetically modified and/or otherwisetreated to correct the dysregulation so that they exhibit normal,non-disease related expression and/or activity levels prior toadministration.

Induced pluripotent stem cells can be produced by expressing acombination of reprogramming factors in a somatic cell. Suitablereprogramming factors that promote and induce iPSC generation includeone or more of Oct4, Klf4, Sox2, c-Myc, Nanog, C/EBPα, Esrrb, Lin28, andNr5a2. In certain embodiments, at least two reprogramming factors areexpressed in a somatic cell to successfully reprogram the somatic cell.In other embodiments, at least three reprogramming factors are expressedin a somatic cell to successfully reprogram the somatic cell. In otherembodiments, at least four reprogramming factors are expressed in asomatic cell to successfully reprogram the somatic cell.

iPSCs may be derived by methods known in the art including the use ofintegrating viral vectors (e.g., lentiviral vectors, induciblelentiviral vectors, and retroviral vectors), excisable vectors (e.g.,transposon and floxed lentiviral vectors), and non-integrating vectors(e.g., adenoviral and plasmid vectors) to deliver the aforementionedgenes that promote cell reprogramming (see e.g., Takahashi and Yamanaka,Cell 126:663-676 (2006); Okita. et al., Nature 448:313-317 (2007);Nakagawa et al., Nat. Biotechnol. 26:101-106 (2007); Takahashi et al.,Cell 131:1-12 (2007); Meissner et al. Nat. Biotech. 25:1177-1181 (2007);Yu et al. Science 318:1917-1920 (2007); Park et al. Nature 451:141-146(2008); and U.S. Pat. Application Publication No. 2008/0233610, whichare hereby incorporated by reference in their entirety). Other methodsfor generating IPS cells include those disclosed in WO2007/069666,WO2009/006930, WO2009/006997, WO2009/007852, WO2008/118820, U.S. Pat.Application Publication Nos. 2011/0200568 to Ikeda et al., 2010/0156778to Egusa et al., 2012/0276070 to Musick, and 2012/0276636 to Nakagawa,Shi et al., Cell Stem Cell 3(5): 568-574 (2008), Kim et al., Nature 454:646-650 (2008), Kim et al., Cell 136(3) :411-419 (2009), Huangfu et al.,Nature Biotechnology 26: 1269-1275 (2008), Zhao et al., Cell Stem Cell3: 475-479 (2008), Feng et al., Nature Cell Biology 11: 197-203 (2009),and Hanna et al., Cell 133(2): 250-264 (2008), which are herebyincorporated by reference in their entirety.

Integration free approaches, i.e., those using non-integrating andexcisable vectors, for deriving iPSCs free of transgenic sequences areparticularly suitable in the therapeutic context. Suitable methods ofiPSC production that utilize non-integrating vectors include methodsthat use adenoviral vectors (Stadtfeld et al., “Induced Pluripotent StemCells Generated without Viral Integration,” Science 322: 945-949 (2008),and Okita et al., “Generation of Mouse Induced Pluripotent Stem Cellswithout Viral Vectors,” Science 322: 949-953 (2008), which are herebyincorporated by reference in their entirety), Sendi virus vectors(Fusaki et al., “Efficient Induction of Transgene-Free Human PluripotentStem Cells Using a Vector Based on Sendi Virus, an RNA Virus That DoesNot Integrate into the Host Genome,” Proc Jpn Acad. 85: 348-362 (2009),which is hereby incorporated by reference in its entirety),polycistronic minicircle vectors (Jia et al., “A Nonviral MinicircleVector for Deriving Hyman iPS Cells,” Nat. Methods 7: 197-199 (2010),which is hereby incorporated by reference in its entirety), andself-replicating selectable episomes (Yu et al., “Human InducedPluripotent Stem Cells Free of Vector and Transgene Sequences,” Science324: 797-801 (2009), which is hereby incorporated by reference in itsentirety). Suitable methods for iPSC generation using excisable vectorsare described by Kaji et al., “Virus-Free Induction of Pluripotency andSubsequent Excision of Reprogramming Factors,” Nature 458: 771-775(2009), Soldner et al., “Parkinson’s Disease Patient-Derived InducedPluripotent Stem Cells Free of Viral Reprogramming Factors,” Cell136:964-977 (2009), Woltjen et al., “PiggyBac Transposition ReprogramsFibroblasts to Induced Pluripotent Stem Cells,” Nature 458: 766-770(2009), and Yusa et al., “Generation of Transgene-Free InducedPluripotent Mouse Stem Cells by the PiggyBac Transposon,” Nat. Methods6: 363-369 (2009), which are hereby incorporated by reference in theirentirety. Suitable methods for iPSC generation also include methodsinvolving the direct delivery of reprogramming factors as recombinantproteins (Zhou et al., “Generation of Induced Pluripotent Stem CellsUsing Recombinant Proteins,” Cell Stem Cell 4: 381-384 (2009), which ishereby incorporated by reference in its entirety) or as whole-cellextracts isolated from ESCs (Cho et al., “Induction of Pluripotent StemCells from Adult Somatic Cells by Protein-Based Reprogramming withoutGenetic Manipulation,” Blood 116: 386-395 (2010), which is herebyincorporated by reference in its entirety).

The methods of iPSC generation described above can be modified toinclude small molecules that enhance reprogramming efficiency or evensubstitute for a reprogramming factor. These small molecules include,without limitation, epigenetic modulators such as the DNAmethyltransferase inhibitor 5′-azacytidine, the histone deacetylaseinhibitor VPA, and the G9a histone methyltransferase inhibitor BIX-01294together with BayK8644, an L-type calcium channel agonist. Other smallmolecule reprogramming factors include those that target signaltransduction pathways, such as TGF-β inhibitors and kinase inhibitors(e.g., kenpaullone) (see review by Sommer and Mostoslavsky,“Experimental Approaches for the Generation of Induced Pluripotent StemCells,” Stem Cell Res. Ther. 1:26 doi:10.1186/scrt26 (2010), which ishereby incorporated by reference in its entirety).

Suitable iPSCs derived from adult fibroblasts can be obtained followingthe procedure described in Streckfuss-Bomeke et al., “Comparative Studyof Human-Induced Pluripotent Stem Cells Derived from Bone Marrow Cells,Hair Keratinocytes, and Skin Fibroblasts,” Eur. Heart J. doi:10.1093/eurheartj/ehs203 (2012), which is hereby incorporated byreference in its entirety). iPSCs derived from umbilical cord bloodcells can be obtained as described in Cai et al., “Generation of HumanInduced Pluripotent Stem Cells from Umbilical Cord Matrix and AmnioticMembrane Mesenchymal Cells,” J. Biol. Chem. 285(15): 112227-11234 (2110)and Giorgetti et al., “Generation of Induced Pluripotent Stem Cells fromHuman Cord Blood Cells with only Two Factors: Oct4 and Sox2,” NatureProtocols, 5(4):811-820 (2010),which are hereby incorporated byreference in their entirety. iPSCs derived from bone marrow cells can beobtained using methods described in Streckfuss-Bomeke et al.,“Comparative Study of Human-Induced Pluripotent Stem Cells Derived fromBone Marrow Cells, Hair Keratinocytes, and Skin Fibroblasts,” Eur. HeartJ. doi: 10.1093/eurheartj/ehs203 (Jul. 12, 2012), and Hu et al.,“Efficient Generation of Transgene-Free Induced Pluripotent Stem Cellsfrom Normal and Neoplastic Bone Marrow and Cord Blood MononuclearCells,” Blood doi: 10.1182/blood-2010-07-298331 (Feb. 4, 2011) which arehereby incorporated by reference in their entirety). iPSCs derived fromperipheral blood can be obtained following the methods described inSommer et al., “Generation of Human Induced Pluripotent Stem Cells fromPeripheral Blood using the STEMCCA Lentiviral Vector,” J. Vis. Exp. 68:e4327 doi:10.3791/4327 (2012), which is hereby incorporated by referencein its entirety. iPS cells contemplated for use in the methods describedherein are not limited to those described in the above references, butrather includes cells prepared by any method as long as the cells havebeen artificially induced from cells other than pluripotent stem cells.

Methods of obtaining highly enriched preparations of oligodendrocyteprogenitor cells from the iPSCs or embryonic stem cells (e.g., humanembryonic stem cells) that are suitable for treating a neuropsychiatricdisorder as described herein are disclosed in WO2014/124087 to Goldmanand Wang, and Wang et al., “Human iPSC-Derived OligodendrocyteProgenitors Can Myelinate and Rescue a Mouse Model of CongenitalHypomyelination,” Cell Stem Cell 12(2):252-264 (2013), which are herebyincorporated by reference in their entirety.

Briefly, oligodendrocyte progenitor cells are derived from a pluripotentpopulation of cells, i.e., iPSCs or embryonic stem cells, using aprotocol that directs the pluripotent cells through serial stages ofneural and glial progenitor cell differentiation. Each stage of lineagerestriction is characterized and identified by the expression of certaincell proteins. Stage 1 of this process involves culturing thepluripotent cell population under conditions effective to induceembryoid body formation. As described herein, the pluripotent cellpopulation may be maintained in co-culture with other cells, such asembryonic fibroblasts, in an embryonic stem cell (ESC) media (e.g.,DMEM/F12 containing a suitable serum replacement and bFGF). Thepluripotent cells are passaged before reaching 100% confluence, e.g.,80% confluence, when colonies are approximately 250-300 µm in diameter.The pluripotential state of the cells is readily assessed using markersto SSEA4, TRA-1-60, OCT-4, NANOG, and/or SOX2.

To generate embryoid bodies (EBs) (Stage 2), which are complexthree-dimensional cell aggregates of pluripotent stem cells, pluripotentcell cultures are dissociated once they achieved ~80% confluence withcolony diameters at or around 250-300 µm. The EBs are initially culturedin suspension in ESC media without bFGF, and then switched to neuralinduction medium supplemented with bFGF and heparin. To induceneuroepithelial differentiation (Stage 3) EBs are plated and cultured inneural induction medium supplemented with bFGF, heparin, laminin, thenswitched to neural induction media supplemented with retinoic acid.Neuroepithelial differentiation is assessed by the co-expression of PAX6and SOX1, which characterize central neural stem and progenitor cells.

To induce pre-oligodendrocyte progenitor cell (“pre-OPCs”)differentiation, neuroepithelial cell colonies are cultured in thepresence of additional factors including retinoic acid, B27 supplement,and a sonic hedgehog (shh) agonist (e.g., purmophamine). The appearanceof pre-OPC colonies is assessed by the presence of OLIG2 and/or NKX2.2expression. While both OLIG2 and NKX2.2 are expressed by centraloligodendrocyte progenitor cells, NKX2.2 is a more specific indicator ofoligodendroglial differentiation. Accordingly, an earlypre-oligodendrocyte progenitor cell stage is marked by OLIG⁺/NKX2.2⁻cell colonies. OLIG⁺/NKX2.2⁻ early pre-OPCs are differentiated intolater-stage OLIG⁺/NKX2.2⁺ pre-OPCs by replacing retinoic acid with bFGF.At the end of Stage 5, a significant percentage of the cells arepre-OPCs as indicated by OLIG2⁺/NKX2.2⁺ expression profile.

Pre-OPCs are further differentiated into bipotential oligodendrocyteprogenitor cells by culture in glial induction media supplemented withgrowth factors such as triiodothyronine (T3), neurotrophin 3 (NT3),insulin growth factor (IGF-1), and platelet-derived growth factor-AA(PDGF-AA) (Stage 6). These culture conditions can be extended for 3-4months or longer to maximize the production of myelinogenicoligodendrocyte progenitor cells when desired. Cell preparationssuitable for transplantation into an appropriate subject are identifiedas containing PDGFRα⁺ oligodendrocyte progenitor cells.

Alternative methods of obtaining preparations of oligodendrocyteprogenitor cells from the iPSCs or embryonic stem cells that are knownin the art can also be used to produce a therapeutic population of cellssuitable for treating a neuropsychiatric disorder as described herein.In yet another embodiment, glial progenitor cells can be extracted fromembryonic tissue, fetal tissue, or adult brain tissue containing a mixedpopulation of cells directly by using the promoter specific separationtechnique, as described in U.S. Pat. Application Publication Nos.20040029269 and 20030223972 to Goldman, which are hereby incorporated byreference in their entirety. In accordance with this embodiment, theglial progenitor cells are isolated from ventricular or subventricularzones of the brain or from the subcortical white matter.

In some embodiments, it may be preferable to enrich a cell preparationcomprising oligodendrocyte progenitor cells to increase theconcentration and/or purity of the therapeutic oligodendrocyteprogenitor cells prior to administration. Accordingly, in oneembodiment, the A2B5 monoclonal antibody (mAb) that recognizes and bindsto gangliosides present on glial progenitor cells early in thedevelopmental or differentiation process can be used to separate glialprogenitor cells from a mixed population of cells (Nunes et al.,“Identification and Isolation of Multipotential Neural Progenitor CellsFrom the Subcortical White Matter of the Adult Human Brain.,” Nat Med.9(4):439-47 (2003), which is hereby incorporated by reference in itsentirety). Using the A2B5 mAb, glial progenitor cells can be separated,enriched, or purified from a mixed population of cell types. In anotherembodiment, selection of CD140α/PDGFRα positive cells is employed toproduce a purified or enriched preparation of bipotential glialprogenitor cells. In another embodiment, selection of CD9 positive cellsis employed to produce a purified or enriched preparation ofoligodendrocyte-biased progenitor cells. In yet another embodiment, bothCD140α/PDGFRα and CD9 positive cell selection is employed to produce apurified or enriched preparation of oligodendrocyte progenitor cells. Ina further embodiment, selection of CD44 positive cells is employed toproduce a purified or enriched preparation of astrocyte-biasedprogenitor cells (Liu et al., “CD44 Expression IdentifiesAstrocyte-Restricted Precursor Cells,” Dev. Biol. 276(1):31-46 (2004),which is hereby incorporated by reference in its entirety.) In anotherembodiment, both CD140α/PDGFRα and CD44 positive cell selection isemployed to produce a purified or enriched preparation ofoligodendrocyte progenitor cells. In another embodiment, CD140α/PDGFRα,CD9, and CD44 positive cell selection is employed to produce a purifiedor enriched preparation of oligodendrocyte progenitor cells.

The administered glial progenitor cell preparation is optionallynegative for a PSA-NCAM marker and/or other neuronal lineage markers,and/or negative for one or more inflammatory cell markers, e.g.,negative for a CD11 marker, negative for a CD32 marker, and/or negativefor a CD36 marker (which are markers for microglia). Optionally, thepreparation of glial progenitor cells is negative for any combination orsubset of these additional markers. Thus, for example, the preparationof glial progenitor cells is negative for any one, two, three, or fourof these additional markers.

In accordance with the method of treating a neuropsychiatric disorder asdescribed herein, the selected preparation of administered glialprogenitor cells comprises at least about 80% glial progenitor cells,including, for example, about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,100% glial progenitor cells. The selected preparation of glialprogenitor cells can be relatively devoid (e.g., containing less than20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of other cells types such asneurons or cells of neuronal lineage, fibrous astrocytes and cells offibrous astrocyte lineage, multipotent cells, and pluripotential stemcells (like ES cells). Optionally, examplary cell populations aresubstantially pure populations of glial progenitor cells.

Positive and/or negative selection for cell markers of interest (e.g.,PDGFRα marker, A2B5 marker, and/or a CD44 marker) can be carried outserially or sequentially and can be performed using conventional methodsknown in the art such as immunopanning. The selection methods optionallyinvolve the use of fluorescence sorting (FACS), magnetic sorting (MACS),or any other method that allows rapid, efficient cell sorting. Examplesof methods for cell sorting are taught for example in U.S. Pat. No.6,692,957 to Goldman, which is hereby incorporated by reference in itsentirety, at least for compositions and methods for cell selection andsorting.

Generally, cell sorting methods use a detectable moiety. Detectablemoieties include any suitable direct or indirect label, including, butnot limited to, enzymes, fluorophores, biotin, chromophores,radioisotopes, colored beads, electrochemical, chemical-modifying orchemiluminescent moieties. Common fluorescent moieties includefluorescein, cyanine dyes, coumarins, phycoerythrin, phycobiliproteins,dansyl chloride, Texas Red, and lanthanide complexes or derivativesthereof.

One of skill in the art readily appreciates how to select for or againsta specific marker. Thus, by way of example, a population of cells sortedfor a particular marker includes identifying cells that are positive forthat particular marker and retaining those cells for further use orfurther selection steps. A population of cells sorted against a specificmarker includes identifying cells that are positive for that particularmarker and excluding those cells for further use or further selectionsteps.

The glial progenitor cell preparations of described herein, includingthe enriched preparations can be optionally expanded in culture toincrease the total number of cells for therapeutic administration. Thecells can be expanded by either continuous or pulsatile exposure toPDGF-AA or AB as mitogens that support the expansion of oligodendrocyteprogenitor cells; they can be exposed to fibroblast growth factors,including FGF2, FGF4, FGF8 and FGF9, which can support the mitoticexpansion of the glial progenitor cells, but which can bias theirdifferentiation to a mixed population of astrocytes as well asoligodendrocytes. The cells can also be expanded in media supplementedwith combinations of FGF2, PDGF, and NT3, which can optionally besupplemented with either platelet-depleted or whole serum (see Nunes etal. “Identification and Isolation of Multipotent Neural Progenitor Cellsfrom the Subcortical White Matter of the Adult Human Brain,” NatureMedicine 9:239-247; Windrem et al., “Fetal and Adult HumanOligodendrocyte Progenitor Cell Isolates Myelinate the CongenitallyDysmyelinated Brain,” Nature Medicine 10:93-97 (2004), which areincorporated by reference for the methods and compositions describedtherein).

In accordance with the methods described herein, the glial progenitorcell population is administered bilaterally into multiple sites of thesubject being treated as described in Han et al., “Forebrain Engraftmentby Human Glial Progenitor Cells Enhances Synaptic Plasticity andLearning Adult Mice,” Cell Stem Cell 12:342-353 (2013) and Wang et al.,“Human iPSCs-Derived Oligodendrocyte Progenitor Cells Can Myelinate andRescue a Mouse Model of Congenital Hypomyelination,” Cell Stem Cell 12:252-264 (2013), which are hereby incorporated by reference in theirentirety). Methods for transplanting nerve tissues and cells into hostbrains are described by Bjorklund and Stenevi (eds), Neural Grafting inthe Mammalian CNS, Ch. 3-8, Elsevier, Amsterdam (1985); U.S. Pat. No.5,082,670 to Gage et al.; and U.S. Pat. No. 6,497,872 to Weiss et al.,which are hereby incorporated by reference in their entirety. Typicalprocedures include intracerebral, intraventricular, intrathecal, andintracisternal administration.

The glial progenitor cell preparation can be delivered directly to theforebrain subcortex, specifically into the anterior and posterioranlagen of the corpus callosum. Glial progenitor cells can also bedelivered to the cerebellar peduncle white matter to gain access to themajor cerebellar and brainstem tracts. Glial progenitor cells can alsobe delivered to the spinal cord.

Alternatively, the cells may be placed in a ventricle, e.g. a cerebralventricle. Grafting cells in the ventricle may be accomplished byinjection of the donor cells or by growing the cells in a substrate suchas 30% collagen to form a plug of solid tissue which may then beimplanted into the ventricle to prevent dislocation of the graft cells.For subdural grafting, the cells may be injected around the surface ofthe brain after making a slit in the dura.

Delivery of the cells to the subject can include either a single step ora multiple step injection directly into the nervous system. Althoughadult and fetal oligodendrocyte precursor cells disperse widely within atransplant recipient’s brain, for widespread neuropsychiatric disorders,multiple injections sites can be performed to optimize treatment.Injection is optionally directed into areas of the central nervoussystem such as white matter tracts like the corpus callosum (e.g., intothe anterior and posterior anlagen), dorsal columns, cerebellarpeduncles, cerebral peduncles. Such injections can be made unilaterallyor bilaterally using precise localization methods such as stereotaxicsurgery, optionally with accompanying imaging methods (e.g., highresolution MRI imaging). One of skill in the art recognizes that brainregions vary across species; however, one of skill in the art alsorecognizes comparable brain regions across mammalian species.

In one embodiment, the oligodendrocyte progenitor cell preparation isinjected as dissociated cells. In another embodiment, theoligodendrocyte progenitor cell preparation is provided asnon-dissociated cells. In either case, the cellular transplantsoptionally comprise an acceptable solution. Such acceptable solutionsinclude solutions that avoid undesirable biological activities andcontamination. Suitable solutions include an appropriate amount of apharmaceutically-acceptable salt to render the formulation isotonic.Examples of the pharmaceutically-acceptable solutions include, but arenot limited to, saline, Hank’s Balanced Salt Solution, Ringer’ssolution, dextrose solution, and culture media. The pH of the solutionis preferably from about 5 to about 8, and more preferably from about 7to about 7.5.

The injection of the dissociated cellular transplant can be a streaminginjection made across the entry path, the exit path, or both the entryand exit paths of the injection device. Suitable injection devicesinclude cannula, needle, insertion tube, cannula guided by an insertiontube. Automation and stereotactic positioning systems can be used toprovide precise delivery to targeted regions with a uniform entry andexit speed and an injection speed and volume.

The number of glial progenitor cells administered to the subject canrange from about 10²-10⁹ at each administration (e.g., injection site),depending on the size and species of the recipient, and the volume oftissue requiring cell replacement. Single administration (e.g.,injection) doses can span ranges of 1 × 10³- 9 × 10³, 1 × 10⁴ - 9 × 10⁴,1 × 10⁵ - 9 × 10⁵, 1 × 10⁶ - 9 × 10⁶, 1 × 10⁷ - 9 × 10⁷, 1 × 10⁸- 9 ×10⁸, 1 × 10⁹ - 9 × 10⁹, 1 × 10³ - 9 × 10³, or any amount in total for atransplant recipient patient. In one embodiment, the administered doseis 1× 10⁷ -4 × 10⁷ cells. To achieve such doses, cell preparation havinga concentration of 1-2 × 10³ cells/µl, 1-2 × 10⁴ cells/µl, 1-2 × 10⁵cells/µl, 1-2 × 10⁶ cells/µl, 1-2 × 10⁷ cells/µl of pharmaceuticallyacceptable carrier are prepared. In one embodiment, the cell preparationfor administration has a concentration of 1 × 10⁵ - 2 × 10⁵ cells/µl ina total volume of about 25 µl to about 50 µl.

Since the CNS is an immunologically privileged site, administered cells,including xenogeneic, can survive and, optionally, no immunosuppressantdrugs or a typical regimen of immunosuppressant agents are used in thetreatment methods. However, optionally, an immunosuppressant agent mayalso be administered to the subject prior to and after receiving thecell therapy. Immunosuppressant agents and their dosing regimens areknown to one of skill in the art and include such agents asAzathioprine, Azathioprine Sodium, Cyclosporine, Daltroban, GusperimusTrihydrochloride, Sirolimus, Mycophenolate mofetil (MMF),and Tacrolimus.In one embodiment, a combination of any of the aforementionedimmunosuppressant agents are administered to the subject. In oneembodiment, a combination of MMF and tacrolimus are administered to thesubject. Dosages ranges and duration of the regimen can be varied withthe disorder being treated; the extent of rejection; the activity of thespecific immunosuppressant employed; the age, body weight, generalhealth, sex and diet of the subject; the time of administration; theroute of administration; the rate of excretion of the specificimmunosuppressant employed; the duration and frequency of the treatment;and drugs used in combination. One of skill in the art can determineacceptable dosages for and duration of immunosuppression. The dosageregimen can be adjusted by the individual physician in the event of anycontraindications or change in the subject’s status.

In one embodiment, one or more immunosuppressant agents are administeredto the subject starting at 10 weeks prior to cell administration. In oneembodiment, the one or more immunosuppressant agents are administered tothe subject starting at 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4weeks, 3 weeks, 2 weeks, 1 week, 7 days, 6 days, 5 days, 4 days, 3 days,2 days, 1 day, < 24 hours prior to cell administration. In oneembodiment, one or more immunosuppressant agents are administered to thesubject starting on the day of cell administration and continuing for 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months post administration. In oneembodiment, the one or more immunosuppressant agents are administered tothe subject for > 1 year following administration.

As used herein, “treating” or “treatment” refers to any indication ofsuccess in amelioration of an injury, pathology, or condition, includingany objective or subjective parameter such as abatement; remission;diminishing of symptoms or making the injury, pathology, or conditionmore tolerable to the patient; slowing the rate of degeneration ordecline; making the final point of degeneration less debilitating; orimproving a subject’s physical or mental well-being. The treatment oramelioration of symptoms can be based on objective or subjectiveparameters; including the results of a physical examination,neurological examination, and/or psychiatric evaluation. “Treating”includes the administration of glial progenitor cells to prevent ordelay, to alleviate, or to arrest or inhibit development of the symptomsor conditions associated with schizophrenia, autism spectrum disorder,bipolar disorder, or any other neuropsychiatric disorder. “Therapeuticeffect” refers to the reduction, elimination, or prevention of thedisease, symptoms of the disease, or side effects of a disease,condition or disorder in the subject. Treatment may be prophylactic (toprevent or delay the onset or worsening of the disease, condition ordisorder, or to prevent the manifestation of clinical or subclinicalsymptoms thereof) or therapeutic suppression or alleviation of symptomsafter the manifestation of the disease, condition or disorder.

A “dosage effective to treat,” as used herein refers to the amount ofcells that is effective for production of a desired result. This amountvaries, for example, depending upon the health and physical condition ofthe individual to be treated, the mental and emotional capacity of theindividual, the degree of protection desired, the formulation, theattending physician’s assessment of the medical situation, and otherrelevant factors.

Another aspect of the present disclosure relates to a method of treatinga neuropsychiatric disorder. This method includes selecting a subjecthaving the neuropsychiatric disorder and administering, to the selectedsubject, a potassium (K⁺) channel activator at a dosage effective torestore normal brain interstitial glial K⁺ levels in the selectedsubject and treat the neuropsychiatric disorder.

Suitable subjects as well as neuropsychiatric disorders suitable fortreatment in accordance with this aspect of the disclosure are disclosedsupra. Exemplary neuropsychiatric disorders to be treated using themethods described herein include, without limitation, schizophrenia,autism spectrum disorders, and bipolar disorder.

As described herein, neuropsychiatric disorders, such as schizophrenia,involve the dysregulated expression of numerous glial progenitor cellgenes that contribute and/or cause impaired glial cell differentiation.In particular, the expression levels of numerous potassium channel genesare significantly downregulated in the disease state. These resultsindicate a role for dysregulated glial potassium channel function andglial potassium levels in neuropsychiatric disorders like schizophrenia,autism disorders, and bipolar disorders. Accordingly, a subject having aneuropsychiatric disorder will benefit therapeutically from theadministration of one or more K⁺ channel activators to restore normal,healthy brain interstitial glial K⁺ levels.

As used herein, a “K⁺ channel” refers to a protein or polypeptide thatis involved in receiving, conducting, and transmitting signals in anexcitable cell. Potassium channels are typically expressed inelectrically excitable cells, including glial cells, and can formheteromultimeric structures, e.g., composed of pore-forming andregulatory subunits. Examples of potassium channels include: (1) thevoltage-gated potassium channels, (2) the inwardly rectifying channels,(3) the tandem pore channels, and (3) the ligand-gated channels. For adetailed description of potassium channels, see Kandel E. R. et al.,Principles of Neural Science, second edition, (Elsevier SciencePublishing Co., Inc., N.Y. (1985)), which is hereby incorporated byreference in its entirety.

Potassium regulation in the central nervous system is mediated by netpotassium uptake and potassium spatial buffering (Kofuji and Newman.,“Regulation of Potassium by Glial Cells in the Central Nervous System,”Springer Science & Business Media (2008), which is hereby incorporatedby reference in its entirety). For K⁺ uptake, excess extracellular K⁺ istaken up and sequestered within glial cells by the action of the Na⁺,K⁺-ATPase, or by K⁺ flux through transporters or K⁺ channels (Kofuji andNewman., “Regulation of Potassium by Glial Cells in the Central NervousSystem,” Springer Science & Business Media (2008), which is herebyincorporated by reference in its entirety). In spatial buffering, K⁺ istransferred from regions of elevated K⁺ concentration to regions oflower K⁺ concentration by a current flow through glial cells (i.e.,glial K⁺ conductance) (see Orkand et al., “Effect of Nerve Impulses onthe Membrane Potential of Glial Cells in the Central Nervous System ofAmphibia,” J Neurophysiol 29:788 -806 (1966), which is herebyincorporated by reference in its entirety).

In accordance with this aspect of the disclosure, the selected subjecthaving the neuropsychiatric disorder has dysregulated glial K⁺ channelfunction characterized by defective glial K⁺ conductance, defectiveglial K⁺ uptake, and/or defective glial K⁺ channel expression.

Several K⁺ channel activators that act either specifically ornon-specifically on K⁺ channels are known in the art and are suitablefor use in the present invention to restore channel activity. Such K⁺activators include, without limitation, ethyl[2-amino-4-[[(4-fluorophenyl)methyl]amino]phenyl]carbamate (retigabine),N-[2-Amino-6-[[4-fluorophenyl)methyl]amino]-3-pyridinyl]carbamic acidethyl ester maleate (flupirtine),N-[3,5-Bis(trifluoromethyl)phenyl]-N′-[2,4-dibromo-6-(2H-tetrazol-5-yl)phenyl]urea(NS 5806), N-(2-Chloro-5-pyrimidinyl)-3,4-difluorobenzamide (ICA 69673),4-Chloro-N-(6-chloro-3-pyridinyl)benzamide (ICA 110381),5-(2-Fluorophenyl)-1,3-dihydro-3-(1H-indol-3-ylmethyl)-1-methyl-2H-1,4-benzodiazepin-2-one(L-364373),N-(2,4,6-Trimethylphenyl)-bicyclo[2.2.1]heptane-2-carboxamide (ML 213),(2R)-N-[4-(4-Methoxyphenyl)-2-thiazolyl]-1-[(4-methylphenyl)sulfonyl]-2-piperidinecarboxamide(ML 277), N,N′-Bis[2-hydroxy-5-(trifluoromethyl)phenyl]urea (NS 1643),N-[4-Bromo-2-(1H-tetrazol-5-yl-phenyl]-N′-[3-(trifluoromethyl)phenyl]-urea(NS 3623),5-(2,6-Dichloro-5-fluoro-3-pyridinyl)-3-phenyl-2-(trifluoromethyl)-pyrazolo[1,5-a]pyrimidin-7(4H)-one(QO58), 2-[[4-[2-(3,4-Dichlorophenyl)ethyl]phenyl]amino]benzoic acid (PD118057),trans-3,4-dihydro-3-hydroxy-2,2-dimethyl-4-(2-oxo-1-pyrrolidinyl)-2H-1-benzopyran-6-carbonitrile(cromakalim), 7-Chloro-3-methyl-2H-1,2,4-benzothiadiazine 1,1-dioxide(diazoxide),(3S,4R)-3,4-dihydro-3-hydroxy-2,2-dimethyl-4-(2-oxo-1-pyrrolidinyl)-2H-1-benzopyran-6-carbonitrile(levcromakalim), 6-(1-Piperidinyl)-2,4-pyrimidinediamine 3-oxide(minoxidil),N-(3,4-Difluorophenyl)-N′-(3-methyl-1-phenyl-1H-pyrazol-5-yl)urea (ML297), N-[2-(Nitrooxy)ethyl]-3-pyridinecarboxamide (nicorandil),N-cyano-N′-(1,1-dimethylpropyl)-N″-3-pyridylguanidine (P1075),(Z)-5-Chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H-indole-1-carboxamide(tenidap),N-[(3S,4R)-6-Cyano-3,4-dihydro-3-hydroxy-2,2-dimethyl-2H-1-benzopyran-4-yl]-N-hydroxyacetamide(Y-26763),N-[(3S,4R)-6-Cyano-3,4-dihydro-3-hydroxy-2,2-dimethyl-2H-1-benzopyran-4-yl]-N-(phenylmethoxy)acetamide(Y-27152),N-(4-Phenylsulfonylphenyl)-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide(ZM 226600), N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide(ICA-27243), ICA-105665, 2-(2,6-dichloranilino) phenylacetic acid(diclofenac) and its structural analogs (e.g., NH6),(3R,4R)-4-[3-(6-methoxyquinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluorophenyl)-prop-2-ynyl]-piperidine-3-carboxylicacid (RPR260243),2-[[2-(3,4-Dichlorophenyl)-2,3-dihydro-1H-isoindol-5-yl]amino]nicotinicacid (PD307243), nitrous oxide, halothane, 17β-estradiol,dithiothreitol, naringin,(3S)-(+)-(5-Chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one(BMS 204352), isoflurane, 2-halogenated ethanols, halogenated methanes,sevoflurane, and desflurane.

In one embodiment of this aspect of the disclosure, the administered K⁺channel activator increases the activity of glial G protein-activatedinward rectifier K⁺ channels. The G-protein activated inward rectifyingpotassium (K⁺) channels, GIRKs, are members of a larger family ofinward-rectifying potassium channels, Kirs. As the name suggests, GIRKchannels can be activated by pertussis toxin-sensitive G-protein-coupledreceptors of the Gi subtype through interactions with the G-protein’sβ/γ subunits1-3. However, GIRK regulation is complex and both positiveand negative modulation has been observed through Gs and Gq GPCRs aswell as via other indirect mechanisms. GIRK regulation by GPCRs isbelieved to be linked to biological effects of a variety of GPCRagonists including opioids, acetylcholine, and the GABA receptoragonist, baclofen.

The GIRK channels are comprised of four subunits, GIRK1-4 (akaKir3.1-3.4), encoded by the genes KCNJ3, KCNJ6, KCNJ9, and KCNJ5,respectively. These four subunits can form homo and heterotetramers withunique biophysical properties, regulation, and distribution. GIRKs arefound widely expressed in the brain with the GIRK½ subunit combinationbeing the most common and widespread within the cortex, hippocampus,cerebellum, and various other brain regions, while other subunitcombinations, such as GIRK¼, show very limited expression in the brain.

Exemplary agents that activate glial G protein-activated inwardrectifier K⁺ channels (either specifically or non-specifically) that aresuitable for use in the treatment of a neuropsychiatric disorderinclude, without limitation, flupirtine, nitrous oxide, halothane,17β-estradiol, dithiothreitol, naringin, and derivatives and analoguesthereof.

In another embodiment, the administered K⁺ channel activator increasesthe activity of glial K⁺ voltage-gated channels. The K⁺ voltage-gated(Kv) family of channels includes, among others: (1) thedelayed-rectifier potassium channels, which repolarize the membraneafter each action potential to prepare the cell to fire again; and (2)the rapidly inactivating (A-type) potassium channels, which are activepredominantly at subthreshold voltages and act to reduce the rate atwhich excitable cells reach firing threshold. In addition to beingcritical for action potential conduction, Kv channels also control theresponse to depolarizing, e.g., synaptic, inputs and play a role inneurotransmitter release. As a result of these activities, voltage-gatedpotassium channels are key regulators of neuronal excitability (HilleB., Ionic Channels of Excitable Membranes, Second Edition, Sunderland, MA: Sinauer, (1992), which is hereby incorporated by reference in itsentirety).

There is tremendous structural and functional diversity within the Kvpotassium channel superfamily. This diversity is generated both by theexistence of multiple genes and by alternative splicing of RNAtranscripts produced from the same gene. Nonetheless, the amino acidsequences of the known Kv potassium channels show high similarity. Allappear to be comprised of four, pore forming α-subunits and some areknown to have four cytoplasmic (β-subunit) polypeptides (Jan L. Y. etal. Trends Neurosci 13:415-419 (1990); Pongs, O. et al. Sem Neurosci7:137-146 (1995), which are hereby incorporated by reference in theirentirety).

Thus, in one embodiment, the administered K⁺ channel activator increasesthe activity of glial A-type voltage-gated K⁺ channels. Exemplary agentsthat activate glial A-type voltage-gated K⁺ channels (eitherspecifically or non-specifically) that are suitable use in the treatmentof a neuropsychiatric disorder include, without limitation,N-[3,5-Bis(trifluoromethyl)phenyl]-N′-[2,4-dibromo-6-(2H-tetrazol-5-yl)phenyl]urea(NS5806) and derivatives and analogues thereof.

In another embodiment, the administered K⁺ channel activator increasesthe activity of glial delayed rectifier K⁺ channels. Exemplary delayedrectifier K⁺ channel activators for use in this aspect of the presentdisclosure include, without limitation, retigabine and derivatives andanalogues thereof.

The K ⁺ channel activator may also affect glial tandem pore domain K⁺channels. Tandem pore domain K⁺ channels comprise a family of 15 membersfrom what is known as “leak channels.” These channels allow the constantpassage of K⁺ and are encoded by KCNK1 and KCNK18.

Thus, in one embodiment, the administered K⁺ channel activator increasesthe activity of glial tandem pore domain K⁺ channels, including thepotassium leak channels encoded by KCNK1 through KCNK18 inclusive.Exemplary agents that activate tandem pore domain K⁺ channels (eitherspecifically or non-specifically) that are suitable for use in thetreatment of a neuropsychiatric disorder include, without limitationhalothane, isoflurane, 2-haolgenated ethanols, halogenated methanes,sevoflurane, and desflurane and derivatives and analogues thereof.

In one embodiment, the K⁺ channel activator is not a Kv7 (KCNQ) K⁺channel activator. In another embodiment, the K⁺ channel activator isspecific for glial KCNQ channels, i.e., a KCNQ channel activator that isselective for or targeted to activating KCNQ channels on glial cells,but does not activate KCNQ channels on non-glial cells.

In accordance with the method of treating a subject having aneuropsychiatric disorder, the K⁺ channel activator can be administeredto the subject either as a free base or as a pharmaceutically acceptableacid addition salt. In the latter case, the hydrochloride salt isgenerally preferred but other salts derived from organic or inorganicacids may be also used. Examples of such acids include, withoutlimitation, hydrobromic acid, phosphoric acid, sulphuric acid, methanesulfonic acid, phosphorous acid, nitric acid, perchloric acid, aceticacid, tartaric acid, lactic acid, succinic acid, citric acid, malicacid, maleic acid, aconitic acid, salicylic acid, thalic acid, embonicacid, enanthic acid, and the like.

The total daily dosage of the K⁺ channel activator that is administeredto a subject should be at least the amount required to prevent, reduceor eliminate one or more of the symptoms associated with theneuropsychiatric disorder. The typical daily dosage will be between 20and 400 mg and, in general, the daily dosage should not exceed 1600 mg.Higher doses are tolerated by some patients and daily dosages of 2,000mg or more may be considered in a subject receiving concomitant drugtreatment with agents that may lower the concentration and half-life ofthe K⁺ channel activator. These dosages are simply guidelines and theactual dose selected for an individual subject will be determined by theattending physician based upon clinical conditions and using methodswell-known in the art. The suitable K⁺ channel activator may be providedin either single or multiple dosage regimens or on an as needed regime.For example, a subject may be administered a K⁺ channel activator daily,weekly, or monthly. Alternatively, the subject may need to beadministered the K⁺ channel activator once, twice, or greater than twicedaily depending on the particular neuropsychiatric condition, the stageor progression of the condition, individual symptoms, and the extent andduration of relief achieved.

Suitable routes of administration of the K⁺ channel activator include,without limitation, subcutaneous, intramuscular, intravenous, orinhalation. Accordingly, suitable dosage forms include powders,aerosols, parenteral aqueous suspensions, solutions and emulsions.Sustained release dosage forms may also be used. The K+ channelactivator may be administered as either the sole active agent or incombination with other therapeutically active drugs used to treat theneuropsychiatric disorder or to reduce the progression of theneuropsychiatric disorder.

Another aspect of the present disclosure relates to a non-human mammalmodel of a neuropsychiatric disorder. This non-human mammal has at least30% of all of its glial cells in its corpus callosum being human glialcells derived from a human patient with a neuropsychiatric disorderand/or at least 5% of all of its glial cells in the white matter of itsbrain and/or brain stem being human glial cells derived from a humanpatient with a neuropsychiatric disorder.

The non-human mammal comprising human glial cells derived from a patientwith a neuropsychiatric disorder exhibits the behavioral characteristicsand phenotypes associated with the neuropsychiatric disorder. Forexample, when the neuropsychiatric disorder is schizophrenia, thenon-human mammal exhibits the behavioral phenotype of schizophreniawhich is characterized by diminished prepulse inhibition, higheranxiety, and social avoidance as described in the Examples herein.

In one embodiment, the human glial cells constitute at least 50%, of allglial cells in the corpus callosum of the non-human mammal. In anotherembodiment, the human glial cells constitute at least 70%, of all glialcells in the corpus callosum of the non-human mammal. In anotherembodiment, the human glial cells constitute at least 90%, of all glialcells in the corpus callosum of the non-human mammal. In one embodiment,at least 10% of all glial cells in the white matter of the non-humanmammal’s brain and/or brain stem are human glial cells. In anotherembodiment, at least 15% of all glial cells in the white matter of thenon-human mammal’s brain and/or brain stem are human glial cells. Inanother embodiment, at least 20% or more of all glial cells in the whitematter of the non-human mammal’s brain and/or brain stem are human glialcells. In another embodiment, at least 50% of all glial cells in thecerebellar white matter are human glial cells.

The human neuropsychiatric disorder specific glial cells of thenon-human mammal model described herein may be derived from any suitablesource of glial cells, such as, for example and without limitation,human induced pluripotent stem cells (iPSCs) derived from a subjecthaving a neuropsychiatric disorder, or from glial progenitor cellsisolated from brain tissue of a subject having a neuropsychiatricdisorder using the methods described above.

In accordance with the method of producing the non-human mammal model ofa human neuropsychiatric disorder, the selected preparation ofadministered human neuropsychiatric disorder specific glial cellscomprise at least about 80% glial cells, including, for example, about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% glial cells. The selectedpreparation of glial cells can be relatively devoid (e.g., containingless than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of other cellstypes such as neurons or cells of neuronal lineage, fibrous astrocytesand cells of fibrous astrocyte lineage, and pluripotential stem cells(like ES cells). Optionally, example cell populations are substantiallypure populations of glial cells.

Another aspect relates to a method of producing non-human mammal modelof a neuropsychiatric disorder with human diseased glial cells replacingnative glial cells in the non-human mammal brain. This method involvesproviding a population of isolated human glial cells derived from apatient with a neuropsychiatric disorder, introducing the population ofisolated human glial cells into multiple locations within the forebrainand/or brain stem of a non-human mammal, and recovering a non-humanmammal with human glial cells replacing native glial cells in the brain.

Methods of making a non-human mammal using human fetal cells aredescribed in U.S. Pat. 7,524,491 to Goldman, and Windrem et al.,“Neonatal Chimerization With Human Glial Progenitor Cells Can BothRemyelinate and Rescue the Otherwise Lethally Hypomyelinated ShivererMouse,” Cell Stem Cell 2:553-565 (2008), which are hereby incorporatedby reference in their entirety. See also U.S. Pat. ApplicationPublication No. US20160317681 to Goldman, which is hereby incorporatedby reference in its entirety.

The non-human mammal can be any neonatal, juvenile, or adult non-humanmammal. Exemplary non-human mammals include, without limitation, mice,rats, guinea pigs and other small rodents, dogs, cats, sheep, goats, andmonkeys. In one embodiment the non-human mammal is a mouse. Suitablestrains of mice commonly used as models of disease include, withoutlimitation, CD-1® Nude mice, NU/NU mice, BALB/C Nude mice, BALB/C mice,NIH-III mice, SCID® mice, outbred SCID® mice, SCID Beige mice, C3H mice,C57BL/6 mice, DBA/2 mice, FVB mice, CB17 mice, 129 mice, SJL mice,B6C3F1 mice, BDF1 mice, CDF1 mice, CB6F1 mice, CF-1 mice, Swiss Webstermice, SKH1 mice, PGP mice, and B6SJL mice.

The human neuropsychiatric disorder specific glial cells can beintroduced into multiple locations within the forebrain and/or brainstem of a non-human mammal. Suitable methods of administration include,without limitation intracerebral, intraventricular, intrathecal, andintracisternal administration. The neuropsychiatric disorder specificglial cells may alternatively be administered via intraparenchymal orintracallosal transplantation. The number of human neuropsychiatricdisorder specific glial cells to be introduced into the non-human mammalcan range between 10³-10⁵ cells.

It is desirable that the non-human mammal host accepts the human glialcells with little or no adverse immune recognition. Therefore, in someembodiments, the non-human mammal is immuno-incompetent,immuno-deficient, or immuno-suppressed.

Immunosuppression can be achieved either through the administration ofimmunosuppressive drugs such as cyclosporin, sirolimus, or tacrolimus,or through strategies employing locally applied immunosuppressants.Local immunosuppression is disclosed by Gruber, Transplantation 54:1-11(1992), which is hereby incorporated by reference in its entirety. U.S.Pat. No. 5,026,365 to Rossini, which is hereby incorporated by referencein its entirety, discloses encapsulation methods also suitable for localimmunosuppression.

As an alternative to employing immunosuppression techniques, methods ofgene replacement or knockout using homologous recombination, as taughtby Smithies et al. Nature 317:230-234 (1985), which is herebyincorporated by reference in its entirety, can be applied to donor glialcells for the ablation of major histocompatibility complex (MHC) genes.Donor glial cells lacking MHC expression would allow for thetransplantation of an enriched glial cell population across allogeneicand perhaps even xenogenic, histocompatibility barriers without the needto immunosuppress the recipient. General reviews and citations for theuse of recombinant methods to reduce antigenicity of donor cells arealso disclosed by Gruber, Transplantation 54:1-11 (1992), which ishereby incorporated by reference in its entirety. Exemplary approachesto reduce immunogenicity of transplants by surface modification aredisclosed in WO92/04033 to Faustman, which is hereby incorporated byreference in its entirety.

Alternatively, the immunogenicity of the transplanted cells may bereduced by using any non-human mammal host that possesses a geneticmutation rendering it immunodeficient. Exemplary animal models includethose having a mutation which disrupts the recombination activating gene2 (Rag2) (Shinkai et al., Cell 68:855-867 (1992), which is herebyincorporated by reference in its entirety) or the Rag1 gene (Mombaertset al., Cell 68:869-877 (1992) and Schultz et al., Transplantation76:1036-42 (2003), which are hereby incorporated by reference in theirentirety). Other immunodeficient animal models useful for practicing theproducing the non-human mammals described herein include any of thesevere combined immunodeficient mice (SCID), having a mutation in thePrkdc gene. Preferred SCID mouse models for use in aspect include theNOD-SCID, the NOD-SCID-IL2rg, and the NOG (NOD-SCID/γc^(null)) mousemodels. Additionally, the Nude mouse models, carrying a mutation in theFoxn1 gene are also useful for producing the non-human mammal model of ahuman neuropsychiatric disorder.

After the population of isolated human neuropsychiatric disorderspecific glial cells is introduced into the forebrain and/or brain stemof the non-human mammal, the non-human mammal is recovered. As usedherein, the term “recovering the non-human mammal” refers to a processor means by which the introduced human glial cells are allowed tofunctionally engraft into the brain of the non-human mammal. Exemplarypercentages of human glial cells present in the white matter and/orcorpus callosum of the brain and brain stem of the recovered non-humanmammal model are described supra.

Another aspect of the present disclosure relates to a method ofidentifying an agent suitable for treating a neuropsychiatric disorderthat involves providing a non-human mammal model of the neuropsychiatricdisorder as described supra and providing a candidate agent. The methodfurther includes administering the candidate agent to the non-humanmammal and assessing, as a result of said administering, the therapeuticpotential of the candidate agent as suitable for treating theneuropsychiatric disorder.

EXAMPLES

The examples below are intended to exemplify the practice of embodimentsof the disclosure but are by no means intended to limit the scopethereof.

Materials and Methods for Examples

Patient identification, protection and sampling. Patients from whichthese lines were derived were diagnosed with disabling degrees ofschizophrenia with onset in early adolescence; all patients and theirguardians were consented by a child psychiatrist (RLF) under an approvedprotocol of Case Western School of Medicine, blinded as to subsequentline designations, and no study investigators had access to patientidentifiers.

iPSC line derivation and production of GPCs. Punch biopsies of the skinwere obtained from patients with juvenile onset schizophrenia (ages 10to 17 years old) and controls (ages 24 to 32 years old). Inducedpluripotent stem cells (iPSC) lines were derived from the patientsamples using an excisable floxed polycistronic hSTEMCCA lentiviralvector. Short tandem repeat (STR)-based DNA fingerprinting was used toconfirm iPSC identity, as a match to original patient or control donor.Additional genotyping was performed using Illumina Omni5 SNP arrays. TheiPSCs were then driven toward a glial progenitor cell (GPC) fate usingpreviously described protocols (Wang et al., “Human iPSC-DerivedOligodendrocyte Progenitor Cells can Myelinate and Rescue a Mouse Modelof Congenital Hypomyelination,” Cell Stem Cell 12:252-264 (2013), whichis hereby incorporated by reference in its entirety). Cells wereharvested between 160-240 DIV, by which time most typically expressedthe bipotential GPC marker PDGFαR/CD140a, while the remainder wereA2B5+/CD140a- astrocytes. The karyotypes of all iPSC lines were assessedduring glial differentiation to ensure genotypic stability of the cellsutilized in all experiments presented here (karyotyping by WiCell,Madison, WI). All iPSCs showed a normal karyotype, except for line 51,which was found to have a balanced Robertsonian translocation ofchromosome 13, an anomaly previously associated with juvenile-onsetschizophrenia (Graw et al., “Isochromosome 13 in a Patient withChildhood-Onset Schizophrenia, ADHD, and Motor Tic Disorder,” MolCytogenet 5:2 (2012), which is hereby incorporated by reference in itsentirety).

Host transplantation. Homozygous shiverer mice (The Jackson Laboratory,Bar Harbor, ME) were crossed with homozygous rag2-null immunodeficientmice (Shinkai et al., “RAG2-Deficient Mice Lack Mature Lymphocytes Owingto Inability to Initiate V(D)J Rearrangement,” Cell 68:855-867 (1992),which is hereby incorporated by reference in its entirety) on the C3hbackground (Taconic, Germantown, NY, USA) to generate shi/shi xrag2^(-/-) myelin-deficient, immunodeficient mice (Windrem et al.,“Neonatal Chimerization with Human Glial Progenitor Cells can bothRemyelinate and Rescue the Otherwise Lethally Hypomyelinated ShivererMouse,” Cell Stem Cell 2:553-565 (2008), which is hereby incorporated byreference in its entirety). In addition, ragl^(-/-) normally-myelinatedimmunodeficient mice (B6.129S7-Rag1^(tmlMom)/J), were obtained from theJackson Laboratory and bred in the lab. Suspensions of single-cells orsmall clusters of hiPSC-derived GPCs were spun down to 100,000 cells/µl.Neonates were anesthetized by cooling, and transplanted bilaterally inthe corpus callosum with a total of 200,000 cells, as described (Windremet al., “Fetal and Adult Human Oligodendrocyte Progenitor Cell IsolatesMyelinate the Congenitally Dysmyelinated Brain,” Nat.Med. 10:93-97(2004), which is hereby incorporated by reference in its entirety). At 3months of age (shi/shi x rag2^(-/-)) or after completion of behavioraltesting at 6-9 months (rag1^(-/-) only), transplanted mice wereanesthetized with pentobarbital, then perfusion fixed with coldHBSS^(+/+) followed by 4% paraformaldehyde (PF) with a 2 hourpost-fixation in cold PF. All procedures were approved by the Universityof Rochester’s Committee on Animal Resources.

Immunolabeling. Brains were cryopreserved, embedded in OCT (Tissue-TekOCT, Sakura Finetek, Torrance, CA) and sectioned at 20 µm, eithersagittally or coronally, on a cryostat. Human cells were identified withmouse antihuman nuclei, clone 235-1 at 1:800 (MAB1281, EMD Millipore,Billerica, MA). Myelin basic protein was labeled with rat anti-MBP at1:25 (Ab7349, Abcam, Cambridge, MA), oligodendrocyte progenitors withanti-human-specific PDGF Receptor α (D13C6, XP® rabbit mAb 5241, 1:300,Cell Signaling Technology), oligodendrocytes with mouseanti-human-specific transferrin (clone HT1/13.6.3, 08691231, MPBiomedicals), astrocytes with anti-human-specific GFAP (SMI 21 at1:1000, Covance, Princeton, NJ). Alexa Fluor secondary antibodies, goatanti- mouse, rat, and rabbit 488, 568, 594, and 647 were used at 1:400(Life Technologies, Carlsbad, CA).

Antibodies and dilutions used Antigen Name Dilution Catalog CompanyhGFAP Mouse anti-human (specific) GFAP 1:500 SMI-21R Covance hNA Mouseanti-human nuclear antigen, cl. 235-1 1:800 MAB1281 Millipore hNG2 Mouseanti-NG2, clone 9.2.27 1:200 MAB2029 Millipore MBP Rat anti-Myelin BasicProtein 1:25 ab7349 Abcam Olig2 Rabbit anti-Olig2 1:500 RA25017Neuromics PDGFRα Rabbit anti-PDGFRα, clone D13C6 1:300 5241S Cell SignITransferrin Mouse anti-human transferrin 1:800 ab9538 Abcam Secondaryantibodies AlexaFluor 568 Goat anti-Mouse IgG (H+L) 1:400 A-11031Invitrogen AlexaFluor 568 Goat anti-Mouse IgG1 1:400 A-21124 InvitrogenAlexaFluor 488 Goat anti-Mouse IgG (H+L) 1:400 A-11029 InvitrogenAlexaFluor 488 Goat anti-Mouse IgG1 1:400 A-21121 Invitrogen AlexaFluor568 Goat anti-Rabbit IgG (H+L) 1:400 A-11036 Invitrogen AlexaFluor 488Goat anti-Rabbit IgG (H+L) 1:400 A-11034 Invitrogen Cy5 Goat anti-Rat1:400 A10525 Invitrogen AlexaFluor 568 Goat anti-Rat IgG (H+L) 1:400A-11077 Invitrogen AlexaFluor 488 Goat anti-Rat IgG (H+L) 1:400 A-11006Invitrogen

Western blots. GPCs derived from CWRU22 and CWRU51 were sorted by FACSfor CD140a at DIV160-200, directly into cell lysis buffer (NP40,Invitrogen, FNN0021) with protease inhibitor (Roche, 183617025) on ice.The insoluble fraction was removed by centrifugation at 12,000 g for 5minutes at 4° C., and the supernatant analyzed for total protein withBCATM Protein Assay Kit (Thermo, 23227). 10 µg sample aliquots wereseparated on 4-12% gradient gels by SDS-PAGE electrophoresis (XCellSureLock, Invitrogen, 071210). Separated protein was transferred to PVDFmembranes, which were blocked with 5% dry milk and incubatedsequentially with a rabbit polyclonal anti-neurexin-1 antisera(Millipore, ABN161-1, 1:1000) at 4° C. overnight, then washed andfollowed serially by a mouse monoclonal anti-ß actin (Abcam, ab173838,1:5000) at RT for 1 h, and anti-mouse and anti-rabbit secondaryantibodies (GE Healthcare, 95107-322 and 95107-328, 1:10000) at RT for 1h. Membranes were visualized by chemiluminescence (Mix ECLTM Reagent, GEHealthcare, RPN2236) through exposure of X-ray film. Experiments wererepeated 3 times, with 3 different sets of cells.

Imaging and Quantitative histology. For mapping the distribution ofhuman nuclei, or photographing gross distribution of myelin at lowpower, whole brain sections were imaged on a Leica LMD 6500. Imaging forphenotypic counts was performed on an Olympus BX51 driven by StereoInvestigator software (MBF, Williston, VT).

Astrocyte morphometrics. Shiverer x rag2-null mice were sacrificed at4.5 months of age and their white matter astrocyte morphologiesassessed. 150 µm thick coronal slices were taken by Vibratome at Bregma-1.0 mm from control (22, 37 and C27) or SCZ (51, 164, 193)hGPC-engrafted mice, incubated in mouse anti-hGFAP for 1 week, then 4hrs in Alexa 568 goat anti-mouse. The slices were mounted on slides andimaged at 100x by confocal (Leica SP8). The images were traced usingNeurolucida 360 (MicroBrightfield, Inc.). Individual astrocytes wereselected from the middle of the corpus callosum at mid-depth so as tocapture cells and their processes in their entirety. Nine cells/brainwere analyzed by Neurolucida with Sholl analysis, as 3 cells/slice and 3slices/brain, taken at 500, 1000, and 1500 µm lateral of the midline.Two or three brains were assessed for each of three lines produced fromseparate patients, for a total of 8 brains and 72 tracedcells/condition, for both CTRL and SCZ-engrafted groups. For Shollanalysis, concentric shells placed at successively increasing diametersof 5 µm were centered on the cell body, and the number of intersectionsbetween cell processes and shells counted (Sholl, DA., “DendriticOrganization in the Neurons of the Visual and Motor Cortices of theCat,” J. Anat. 87:387-406 (1953), which is hereby incoporated byreference in its entirety). For the assessment and quantitativedescription of astrocytic fiber 3D architecture, Fan-in analysis (MBFBiosciences) was used as previously described for studies of dendritictopology (Dang et al., “Formoterol, a Long-Acting Beta2 AdrenergicAgonist, Improves Cognitive Function and Promotes Dendritic Complexityin a Mouse Model of Down Syndrome,” Biol. Psychiatry 75:179-188 (2014),which is hereby incorporated by reference in its entirety).

Myelin luminance analysis. To measure forebrain myelination, luminanceanalysis based on measurement of myelin basic protein (MBP)immunofluorescence. Evenly-spaced and uniformly sampled coronal sectionswere stained for MBP as described, and images taken at 10x using a NikonNi-E and Nikon DS-Fi1 camera. The corpus callosum was selected as regionof interest, and mean intensity values were obtained using NIS Elementsv.4.5.

Behavior. Behavioral tests were scored using either ANY-maze (Stoelting,Wood Dale, IL) or EthoVision (Noldus). Behavioral testing began ateither 25 weeks (for pre-pulse inhibition) or 30-36 weeks (all othertests), and typically lasted 3 weeks; starting age was matched betweenexperimentals and controls. A total of 6-12 recipient mice wereengrafted and tested per cell line, or 17-36 mice per group for eachbehavioral comparison, with a roughly equal balance of male (M) andfemale (F) recipients. Tests were performed in the same sequence for allmice, and included: 1) Elevated Plus Maze. Each test mouse was placed inthe center of a raised, plus-shaped apparatus, consisting of 2 enclosedarms and 2 open arms, facing an open arm (Walf et al., “The Use of theElevated Plus Maze as an Assay of Anxiety-Related Behavior in Rodents,”Nat Protoc 2:322-328 (2007), which is hereby incorporated by referencein its entirety). Each tested mouse was videotaped and scored for timespent in the open vs. closed arms. 2) Three chamber social choice. Thetest apparatus is a plexiglass enclosure divided into thirds withconnecting doors (Ugo Basile, Italy) (Yang et al., “AutomatedThree-Chambered Social Approach Task for Mice,” Curr Protoc Neurosci,Chapter 8, Unit 8, 26 (2011), which is hereby incorporated by referencein its entirety). Each test mouse was first acclimated to the centralchamber or 5 minutes. The doors to the outer chambers were then removed,and the test mouse allowed to explore all three chambers for 10 minutes.The test mouse was then guided back to the central chamber, and a samesex and age stranger mouse was placed in a cylindrical container in oneside chamber, while an empty cylindrical container was placed in theopposite side chamber. The mouse was then recorded for 10 minutes, andscored with respect to the amount of time it spent with the strangermouse vs. the empty compartment. 3) Novel Object Recognition. Each testmouse was placed in an empty 1 ft² testing chamber for 5 minutes toacclimate, then removed, and two identical objects were placed in thechamber. The mouse was returned to the chamber with the objects, placedfacing directly away from them, recorded for 10 minutes and scored fortime spent in proximity to each object (Bevins et al., “ObjectRecognition in Rats and Mice: A One-Trial Non-Matching-to-SampleLearning Task to Study ‘Recognition Memory’,” Nat Protoc 1:1306-1311(2006), which is hereby inorporated by reference in its entirety). Afterone hour, the experiment was repeated, with one of the two objectsreplaced by a novel object. 4) Pre-pulse inhibition. Each mouse wasplaced in a restraint chamber inside a larger isolation cabinet,equipped with sound, light, and air puff generators (SR-LAB, San DiegoInstruments), and auditory PPI assessed as described (Geyer et al.,“Measurement of Startle Response, Prepulse Inhibition, and Habituation,”Curr Protoc Neurosci, Chapter 8, Unit 8, 7 (2001), which is herebyincorporated by reference in its entirety). 5) Sucrose preference. Thisexperiment was always performed last, as mice were individually housedin order to measure liquid consumption. Sucrose preference wasdetermined by the percentage of sucrose water consumed as a proportionof all water consumed (Willner et al., “Reduction of Sucrose Preferenceby Chronic Unpredictable Mild Stress, and its Restoration by a TricyclicAntidepressant,” Psychopharmacology (Berl) 93:358-364 (1987), which ishereby incorporated by reference in its entirety). Water is delivered inthe colony by Hydropac (Lab Products, Inc.), so an additional Hydropaccontaining sucrose water was added to the cage and the two packs wereweighed daily.

Activity and sleep assessment. Individually-housed mice were videorecorded in 12″ × 12″ × 13.5” acrylic chambers, using infra-red camerasduring the dark phase, for 72 continuous hours under 12/12 light/darkconditions. The distance traveled in meters per hour was calculated byNoldus Ethovision software, and averaged across 8 CTRL mice (gray fill,lines 22 and 17) and 10 SCZ mice (purple fill, line 52). In addition,transitions between phases of the light cycle (measured 30 minutesbefore and 30 minutes after light changes) were analyzed in terms of thenumber of consecutive seconds of immobility as a percentage of totalimmobility (AnyMaze, Stoelting), per 30 min measurement block asdescribed (McShane et al., “Characterization of the Bout Durations ofSleep and Wakefulness,” J. Neurosci. Methods 193:321-333 (2010); Pack etal., “Novel Method for High-Throughput Phenotyping of Sleep in Mice,”Physiol. Genomics 28:232-238 (2007), which are are hereby incorporatedby reference in their entirety).

Statistical analysis. Unless otherwise noted, analyses were done inGraphPad Prism v.7. Individual tests were performed as noted for eachexperiment. All data are presented as mean ± SEMs.

RNA-seq and bioinformatics. hGPCs assessed for gene expression werefirst sorted by fluorescence-activated cell sorting on the basis of thecell surface marker CD140a (BD Pharmingen) as described (FIG. 3 ) (Simet al., “CD140a Identifies a Population of Highly Myelinogenic,Migration-Competent and Efficiently Engrafting Human OligodendrocyteProgenitor Cells,” Nature Biotechnology 29:934-941 (2011), which ishereby incorporated by reference in its entirety). UsingpolyA-selection, mRNA was isolated from these PDGFRα+ hGPCs, which werederived from iPSCs made from 4 patients with juvenile-onsetschizophrenia (SCZ line numbers 8 [n=4 independent cell preparations],29 [n=3], 51 [n=7], and 164 [n=8]); and 3 demographically similarhealthy controls (CTR lines 22 [n=3], 37 [n=4], and 205 [n=7]).Sequencing libraries were prepared using the TruSeq RNA v2 kit, andsequenced on an Illumina HiSeq 2500 platform for approximately 45million 1×100 bp reads per sample. The sequencing reads werepre-processed by trimming off adapter and low-quality sequences from the3′ end using Trimmomatic (Bolger et al., “Trimmomatic: A FlexibleTrimmer for Illumina Sequence Data,” Bioinformatics 30:2114-2120 (2014),which is hereby incorporated by reference in its entirety). The qualityof reads before and after pre-processing was assessed with FastQC(D′Antonio et al., “RAP: RNA-Seq Analysis Pipeline, A New Cloud-BasedNGS Web Application,” BMC Genomics 16:S3 (2015), which is herebyincorporated by reference in its entirety), and the pre-processed readswere then aligned to the RefSeq NCBI reference human genome versionGRCh38 (Pruitt et al., “NCBI Reference Sequences (RefSeq): A CuratedNon-Redundant Sequence Database of Genomes, Transcripts and Proteins,”Nucleic Acids Research 35:D61-65 (2007), which is hereby incorporated byreference in its entirety) with Subread read aligner (Liao et al., “TheSubread Aligner: Fast, Accurate and Scalable Read Mapping bySeed-and-Vote,” Nucleic Acids Research 41:e108 (2013), which is herebyincorporated by reference in its entirety) using Hamming distance tobreak ties between more than one optimal mapping locations. Raw genecounts were obtained from BAM alignment files with the featureCountstool (Liao et al., “Feature Counts: An Efficient General Purpose Programfor Assigning Sequence Reads to Genomic Features,” Bioinformatics30:923-930 (2014), which is hereby incorporated by reference in itsentirety). After eliminating lowly expressed transcripts with a count <5reads in more than 5 samples across the dataset, the count data wasnormalized using the RUVSeq (Risso et al., “Normalization of RNA-SeqData Using Factor Analysis of Control Genes or Samples,” Nat Biotechnol32:896-902 (2014), which is hereby incorporated by reference in itsentirety) R Bioconductor package (Gentleman et al., “Bioconductor: OpenSoftware Development for Computational Biology and Bioinformatics,”Genome Biology 5:R80 (2004), which is hereby incorporated by referencein its entirety) to account for variance. As described in the RUVSeqmanual, the normalization was accomplished in the following three-stepprocedure: 1) in silico negative control genes were determined byfirst-pass differential expression analysis by the edgeR (Robinson etal., “EdgeR: A Bioconductor Package for Differential Expression Analysisof Digital Gene Expression Data,” Bioinformatics 26:139-140 (2010),which is hereby incorporated by reference in its entirety) and DESeq2(Love et al., “Moderated Estimation of Fold Change and Dispersion forRNA-Seq Data with DESeq2,” Genome Biology 15:550 (2014), which is herebyincorporated by reference in its entirety) R Bioconductor packages,taking genes with FDR-adjusted P values >0.75, as calculated by bothmethods (approximately 7000 genes were unaffected by the condition ofinterest); 2) the negative control genes were then used in the RUVsfunction of the RUVSeq package, for calculation of variance factors;and, 3) the second-pass differential expression analysis (5% FDR andlog2 fold change >1) for determining disease-dysregulated genes wasperformed using the original counts, adjusting for RUVs-calculatedvariance factors by multi-factor GLM models implemented in the edgeR andDESeq2 packages.

This three-step analysis, with filtering for low-expressed transcripts,was used to compare each SCZ-derived hGPC cell line to the pooledCTR-derived hGPCs. The intersection of the resulting 4 individual listsof differentially expressed genes was taken as the conservedrepresentative list of SCZ-dysregulated genes. In the normalizationprocedure for each comparison, the number of RUVs-calculated variancefactors was limited to 1 for line 29, 3 for lines 8 and 164, and 7 forline 51, as determined by principal component and hierarchicalclustering analyses performed with native R functions. To obtain averagefold changes and P values for dysregulated genes in all 4 SCZ hGPClines, a differential expression comparison of pooled SCZ to pooled CTRlines was performed by the same filtering and analysis workflow with thenumber of variance factors limited to 9.

For all differential expression comparisons, only the significantresults that agreed between edgeR and DESeq2 methods were used indownstream analysis. Once individual fold changes and P values fordysregulated genes in all 4 SCZ hGPC lines were established relative tocontrol lines, the differential expression of pooled SCZ to pooled CTRlines was performed. For each SCZ cell line, separate DE comparisonswere performed against each control line and the intersection of the DEgenes was taken as a representative list for that SCZ line against thecontrol population. Fold changes and FDR-adjusted P values reported werecalculated by edgeR. Functional annotation of the conserved set ofSCZ-dysregulated genes was done using ToppCluster (Kaimal et al.,“ToppCluster: A Multiple Gene List Feature Analyzer for ComparativeEnrichment Clustering and Network-Based Dissection of BiologicalSystems,” Nucleic Acids Research 38:W96-102 (2010), which is herebyincorporated by reference in its entirety) and Ingenuity PathwayAnalysis (IPA). Network visualization and analysis of the results offunctional annotation were performed in Gephi (Jacomy et al.,“ForceAtlas2, A Continuous Graph Layout Algorithm for Handy NetworkVisualization Designed for the Gephi Software,” PloS one 9:e98679(2014), which is hereby incorporated by reference in its entirety) graphvisualization software.

For streamlined execution of the above data processing and analysisroutines, a set of Python and R scripts was developed. All genomic datahave been deposited to GEO, accession number GSE86906.

Real-Time PCR. Expression levels in SCZ- and control derived GPCs ofselected target genes identified by RNA-seq were assayed by TaqMan LowDensity Array (TLDA) Real-Time PCR. The raw data were analyzed inEspressionSuite Software version 1.1 supplied by Applied Biosystems andexported into HTqPCR R package (Chambers et al., “Highly EfficientNeural Conversion of Human ES and iPS cElls by Dual Inhibition of SMADSignaling,” Nat Biotechnol 27:275-280 (2009), which is herebyincorporated by reference in its entirety) for relative quantificationanalysis.

Example 1 - Generation of iPSCs From Patients With Juvenile-OnsetSchizophrenia

Patients with juvenile-onset schizophrenia, as well as healthy youngadult controls free of known mental illness, were recruited and skinbiopsies were obtained from each. Patient identifiers were not availableto investigators besides the treating psychiatrist, although age,gender, race, diagnosis and medication history accompanied cell lineidentifiers. Fibroblasts were then isolated from each sample; fromthese, 11 new independent hiPS cell lines were derived from 8 patientsamples (5 juvenile-onset schizophrenia patients and 3 healthygender-matched and age-analogous controls (Table 1).

TABLE 1 Subject number hiPSC Line(s) Age of subject Gender EthnicityRNA-Seq of CD140a⁺ GPCs Anatomic assessment N=shiverer mice Behavioralassessment N=myelin w/t mice Control Subjects Cntrl 1 19, 22 26 M C √ √√ Cntrl 2 37 32 F AA √ √ √ Cntrl 3 205 25 M C √ √ √ Cntrl 4 C27 NA NA NA√ √ Schizophrenic Subjects SCZ 1 8 10 F C √ √ SCZ 2 51, 52 16 M C √ √ √SCZ 3 29, 31 12 M C √ √ SCZ 4 164 14 F AA √ √ SCZ 5 193 15 F NA √ √Patients and cell lines used in this study. A total of 11 newindependent iPS cell lines were derived from 8 subjects; 5juvenile-onset schizophrenic patients and 3 healthy controls; anestablished control line (C27) from an additional normal subject waspublished previously (Wang et al., “Human iPSC-Derived OligodendrocyteProgenitor Cells can Myelinate and Rescue a Mouse Model of CongenitalHypomyelination,” Cell Stem Cell 12:252-264 (2013); Chambers et al.,“Highly Efficient Neural Conversion of Human ES and iPS cElls by DualInhibition of SMAD Signaling,” Nat Biotechnol 27:275-280 (2009), whichare hereby incorporated by reference in their entirety). hGPCs derivedfrom these cells were assigned to individual experiments as noted. C,Caucasian; AA, African-American; NA, not available.

iPSC were generated using excisable floxed polycistronic hSTEMCCAlentivirus (Zou et al., “Establishment of Transgene-Free InducedPluripotent Stem Cells Reprogrammed from Human Stem Cells of ApicalPapilla for Neural Differentiation,” Stem Cell Res Ther 3:43 (2012);Somers et al., “Generation of Transgene-Free Lung Disease-Specific HumanInduced Pluripotent Stem Cells Using a Single Excisable Lentiviral StemCell Cassette,” Stem Cells 28:1728-1740 (2010), which are herebyincorporated by reference in their entirety) encoding Oct4, Sox2, Klf4and c-Myc (Takahashi et al., “Induction of Pluripotent Stem Cells fromAdult Human Fibroblasts by Defined Factors,” Cell 131:861-872 (2007);Welstead et al., “Generating iPS Cells from MEFS Through ForcedExpression of Sox-2, Oct-4, c-Myc, and Klf4,” J Vis Exp (2008), whichare hereby incorporated by reference in their entirety). All lines wereinitially characterized and validated as pluripotent using globaltranscriptome profiling by RNA sequencing to assess pluripotent geneexpression, as well as immunostaining for Oct4, Nanog, and SSEA4. Theidentity of each iPSC line was confirmed to match the parental donorfibroblasts using short tandem repeat (STR)-based DNA fingerprinting.iPSC line isolates were also karyotyped concurrently with theseexperiments to confirm genomic integrity. An additionalwell-characterized hiPSC control line, C27 (Chambers et al., “HighlyEfficient Neural Conversion of Human ES and iPS Cells by Dual Inhibitionof SMAD Signaling,” Nat Biotechnol 27:275-280 (2009), which is herebyincorporated by reference in its entirety), was also used, to ensurethat the control engraftment and differentiation data were consistentwith prior studies (Wang et al., “Human iPSC-Derived OligodendrocyteProgenitor Cells Can Myelinate and Rescue a Mouse Model of CongenitalHypomyelination,” Cell Stem Cell 12:252-264 (2013), which is herebyincorporated by reference in its entirety). Altogether, hGPCpreparations were evaluated from 7 iPSC lines derived from 5 SCZpatients, and 5 iPSC lines derived from 4 control subjects (Table 1).The iPSC cells were then instructed to GPC fate as previously described(Wang et al., “Human iPSC-Derived Oligodendrocyte Progenitor Cells CanMyelinate and Rescue a Mouse Model of Congenital Hypomyelination,” CellStem Cell 12:252-264 (2013), which is hereby incorporated by referencein its entirety), and after ≥105 days in vitro (DIV) under glialdifferentiation conditions, validated the predominant GPC phenotype ofeach cell population using flow cytometry for CD140a/PDGFαR (FIG. 2 )(Sim et al., “CD140a Identifies a Population of Highly Myelinogenic,Migration-Competent and Efficiently Engrafting Human OligodendrocyteProgenitor Cells,” Nature Biotechnology 29:934-941 (2011), which ishereby incorporated by reference in its entirety). To optimize glialdifferentiation in vivo, transplants were limited to those preparationsin which most cells were CD140a+ GPCs, with the remainder astroglial.

It was first asked whether SCZ hGPCs differed from wild-type hGPCs inmyelination competence. To this end, SCZ hGPCs were implanted intoneonatal immunodeficient shiverer mice (rag2^(-/-) x MBP^(shi/shi)), acongenitally hypomyelinated mutant lacking myelin basic protein (MBP)(Rosenbluth, J., “Central Myelin in the Mouse Mutant Shiverer,” J CompNeurol 194:639-648 (1980); Roach et al., “Characterization of ClonedcDNA Representing Rat Myelin Basic Protein: Absence of Expression inBrain of Shiverer Mutant Mice,” Cell 34:799-806 (1983), which are herebyincorporated by reference in their entirety). As these otherwisemyelin-deficient mice matured, their engrafted hGPCs differentiated intoboth astrocytes and myelinogenic oligodendrocytes yielding mice chimericfor individual patient-derived glia (Windrem et al., “NeonatalChimerization with Human Glial Progenitor Cells Can Both Remyelinate andRescue the Otherwise Lethally Hypomyelinated Shiverer Mouse,” Cell StemCell 2:553-565 (2008); Windrem et al., “A Competitive Advantage byNeonatally Engrafted Human Glial Progenitors Yields Mice Whose Brainsare Chimeric for Human Glia,” The Journal of Neuroscience: The OfficialJournal of the Society for Neuroscience 34:16153-16161 (2014), which arehereby incorporated by reference in their entirety). By this means, micewith patient-specific, largely humanized forebrain white matter, derivedfrom SCZ or control subjects were established (FIGS. 3A-3D).

Example 2 - SCZ Glial Chimeric Mice Were Uniformly Hypomyelinated

It was first noted that the SCZ hGPCs manifested an aberrant pattern ofmigration upon neonatal transplantation. Normal control hGPCs invariablyexpanded through the white matter before colonizing the cortical graymatter (FIG. 3A), as was previously noted in both fetal tissue- andhiPSC GPC-engrafted shiverer mice (Windrem et al., “NeonatalChimerization with Human Glial Progenitor Cells Can Both Remyelinate andRescue the Otherwise Lethally Hypomyelinated Shiverer Mouse,” Cell StemCell 2:553-565 (2008); Wang et al., “Human iPSC-Derived OligodendrocyteProgenitor Cells Can Myelinate and Rescue a Mouse Model of CongenitalHypomyelination,” Cell Stem Cell 12:252-264 (2013), which are herebyincorporated by reference in their entirety). In contrast, SCZ GPCspreferentially migrated earlier into the gray matter in shiverer mice,with large numbers traversing without stopping in the callosal whitematter (n=4 lines from 4 different patients, each with >3 mice/patient,each vs. paired controls) (FIG. 3B and FIG. 4 ). This resulted insignificantly fewer donor hGPCs in the white matter of shiverersengrafted with SCZ GPCs (FIGS. 3H-3I and FIG. 4 ). Importantly, this wasassociated with substantially diminished central myelination in thesemice, as reflected by both MBP immunostaining (FIGS. 3C-3D and 3E-3F)and myelin luminance (FIG. 3G).

Since the SCZ hGPC-engrafted shiverers manifested deficient myelination,it was asked whether this was due to a relative failure of SCZ hGPCs toremain within white matter, or rather to a cell-intrinsic failure inmyelinogenesis. Examining 19 wk-old SCZ and control hGPC-engraftedshiverer mice, significantly fewer human nuclear antigen (hNA)-defineddonor-derived cells were found in SCZ hGPC-engrafted shiverer whitematter (40,615 ± 2,189 × 10³ hNA⁺ cells/mm3′ n=18) than in miceidentically transplanted with control hGPCs (69,970 ± 4,091/mm³; n=32;p<0.0001 by 2-tailed t test (Fagerland et al., “Performance of fiveTwo-Sample Location Tests For Skewed Distributions with UnequalVariances,” Contemp Clin Trials 30:490-496 (2009); Merman, D. W., “ANote on Preliminary Tests of Equality of Variances,” Br J Math StatPsychol 57:173-181 (2004), which are hereby incorporated by reference intheir entirety) (FIG. 3H). Moreover, the numbers of hNA+ donor cellsco-expressing the oligodendroglial lineage marker Olig2 were similarlydepressed in the SCZ hGPC-engrafted mice (33,619 ± 2,435/mm³, n=26),relative to control hGPC-engrafted mice (46,139 ± 2,858/mm³, n=17;p<0.002) (FIG. 3I). On that basis, it was next found that the density oftransferrin-defined human oligodendroglia was similarly lower in thecallosal white matter of SCZ hGPC chimeras, than in control hGPCchimeras (8,778±892.2/mm³, n=25; vs. 17,754±2,023/mm³, n=17,respectively; p=0.0006, Mann-Whitney) (FIG. 3J). These data indicatethat SCZ GPCs are deficient not only in their colonization of theforebrain white matter, but also in their oligodendrocyticdifferentiation, with a resultant suppression of central myelinogenesis.Together, these findings suggest that SCZ hGPCs migrate aberrantly,traversing rather than homing to developing white matter, thus yieldingrelatively poor white matter engraftment, deficient myelin formation,and premature cortical entry relative to normal GPCs.

Example 3 - SCZ Glial Chimeric Mice Manifested Developmentally-DelayedAstrocytic Maturation

It was next asked whether the SCZ hGPCs that prematurely entered thegray matter differentiated instead into astrocytes in that environment,or whether they rather manifested an impairment in lineage progressionthat prevented their astrocytic differentiation as well. Both SCZ andcontrol hGPC-engrafted shiverer brains were immunostained for astrocyticglial fibrillary acidic protein (GFAP) at 19 weeks after neonatal graft,using a species-specific anti-human GFAP antibody. It was found thatastrocytic maturation from engrafted hGPCs was markedly deficient in theSCZ hGPC-engrafted brains (n=19, derived from 3 SCZ patient lines, andn=12 control mice, from 3 control patients) (FIGS. 5A-5B). In thecallosal white matter, as well as in both the striatal and cortical graymatter, astrocytic differentiation by SCZ hGPCs was significantly lessthan that of control GPCs, such that whereas all control hGPC forebrainsshowed dense human GFAP⁺ astrocytic maturation, far fewer SCZ hGPCsmanifested hGFAP expression and astrocytic phenotype (controls: 6,616 ±672.3 GFAP⁺ cells/mm³ in callosum, n=12; SCZ: 1, 177 ± 276.6 GFAP⁺callosal cells/mm³, n=19; p<0.0001 by 2-way t-test (FIG. 5C). Thisdefect in astrocytic differentiation was consistently observed in allmice (n=19) derived from the 3 SCZ patients assessed, compared to thecontrol GPC-engrafted mice (n=12) derived from 3 normal subjects (FIG.5D), and reflected in part the lower proportion of GFAP⁺ astrocytes thatdeveloped among engrafted human cells in the SCZ HGPC-engrafted brains(FIG. 5E). Furthermore, Sholl analysis of individual astroglialmorphologies (Sholl, D.A., “Dendritic Organization in the Neurons of theVisual and Motor Cortices of the Cat,” J. Anat. 87:387-406 (1953), whichis hereby incorporated by reference in its entirety), as imaged in 150µm sections and reconstructed in Neurolucida (FIG. 5J), revealed thatastrocytes in SCZ hGPC chimeras differed significantly from theircontrol hGPC-derived counterparts, with fewer primary processes (FIG.5F), less proximal branching (FIG. 5G), longer distal fibers (FIG. 5H),and less coherent domain structure (FIG. 5I). Thus, SCZ hGPCs derivedfrom multiple patients exhibited a common defect in phenotypicmaturation, and hence proved deficient in astrocytic differentiation aswell as myelination.

Example 4 - SCZ hGPCs Showed Cell-Autonomous Misexpression ofDifferentiation-Associated Genes

To better define the molecular basis for the apparent impediment toterminal glial differentiation in SCZ GPC-engrafted mice, and to definewhich aspects of that deficit might be cell-autonomous, RNA-seq analysiswas used to identify the differentially expressed genes of SCZiPSC-derived GPCs, relative to those of control-derived glia. Sequencingdata was used to reconstruct the transcriptional patterns of hGPCsderived from 4 different SCZ and 3 control patients. hGPCs were derivedat time points ranging from 154 to 242 days in vitro, and sorted forhGPCs using CD140a-targeted FACS. Using a 5% FDR and a fold-changethreshold of 2, a total of 118 mRNAs were identified that weredifferentially expressed by CD140a-sorted SCZ hGPCs relative to theircontrol iPSC hGPCs (FIGS. 6A-6B). Among those genes most differentiallyexpressed by CD140a-sorted SCZ hGPCs were a host of glialdifferentiation-associated genes, in particular those associated withearly oligodendroglial and astroglial lineage progression, which wereuniformly down-regulated in the SCZ hGPCs relative to their normalcontrols (FIGS. 6C and 6F). These included a coherent set of the key GPClineage transcription factors OLIG1, OLIG2, SOX10, and ZNF488, as wellas genes encoding stage-regulated proteins involved in myelination suchas GPR17, UGT8, OMG, and FA2H (FIG. 6G; see Table 2 and FIG. 7 fordetailed gene expression data).

TABLE 2 Significantly dysregulated genes in SCZ-derived relative tocontrol-derived OPCs (list 1 of 5) Gene ID Log2 FC P Value Entrez GeneName Transcription regulators, & nucleus-associated (10 genes) SLFN 132.832 2.18E-13 schlafen family member 13 SLFN11 2.193 6.47E-09 schlafenfamily member 11 NLRP2 -7.208 4.24E-58 NLR family, pyrin domaincontaining 2 SOX10 -4.448 8.37E-19 SRY-box 10 NROB1 -3.321 5.92E-27nudear receptor subfamily 0 group B member 1 OLIG2 -3.196 4.23E-19oligodendrocyte lineage transcription factor 2 OLIG1 -3.146 5.72E-22oligodendrocyte transcription factor 1 ZNF439 -2.139 6.14E-08 zincfinger protein 439 IRX1 -1.815 5.52E-06 iroquois homeobox 1 ZNF488-1.464 4.44E-07 zinc finger protein 488 Glial differentiation (5 genes)SOX10 -4.448 8.37E-19 SRY-box 10 OLIG2 -3.196 4.23E-19 oligodendrocytelineage transcription factor 2 OLIG1 -3.146 5.72E-22 oligodendrocytetranscription factor 1 DLL3 -2.352 2.09E-24 delta-like 3 (Drosophila)MPZ -2.105 7.66E-13 myelin protein zero Myelination-associated (12genes) SOX10 -4.448 8.37E-19 SRY-box 10 GPR17 -3.357 1.94E-10 Gprotein-coupled receptor 17 UGTB -3.250 4.36E-09 UDP glycosyltransferase8 OLIG2 -3.196 4.23E-19 oligodendrocyte lineage transcription factor 2GAL3ST1 -2.681 4.01E-12 galacrose-3-O-sulfutransferase 1 CNTN1 -2.6755.65E-15 contactin 1 PLLP -2.581 1.21E-24 plasmolipin OMG -2.5615.09E-12 oligodendrocyte myelin glycoprotein FA2H -2.440 4.45E-08 fattyacid 2-hydroxylase SLC8A3 -2.224 2.00E-12 solute carrier family 8(sodium/calcium exchanger), member 3 MPZ -2.105 7.66E-13 myelin proteinzero ZNF488 -1.464 4.44E-07 zinc finger protein 488 Wnt signaling (4genes) WNT7B -2.626 1.46E-07 wingless-type MMTV integration site familymember 7B PCDH15 -2.530 1.42E-10 protocadherin-related 15 PCDH11X -2.3641.25E-08 protocadherin 11 X-linked CDH10 -1.646 9.33E-07 cadherin 10(continued on next page) SLFN13 2.832 2.18E-13 schlafen family member 13SLFN11 2.193 6.47E-09 schlafen family member 11 HS3ST4 2.149 1.44E-04heparan sulfate-glucosamme 3-sulfotransferase 4 ALOX5 1.777 2.35E-05arachidonate 5-lipoxygenase- CA10 -3.550 3.07E-08 carbonic anhydrase XNEU4 -3.361 8.31E-40 neuraminidase 4 (salidase) UGTB -3.250 4.36E-09 UDPglycosyltransferase 8 GAL3ST1 -2.681 4.01E-12galactose-3-0-sulfotransferase 1 CNTN 1 -2.675 5.65E-15 contactin 1CSMD3 -2.560 5.78E-10 CUB and Sushi multiple domains 3 GALNT13 -2.4672.80E-11 polypeptide N-acetylgalactosaminyltransferase 13 FA2H -2.4404.45E-08 fatty acid 2-hydroxylase AOAH -2.271 2.99E-12 acyloxyacylhydrolase KIF19 -1.644 5.10E-06 kinesin family member 19 DSEL -1.1511.35E-10 dermatan sulfate epimerase-like ALOXS 1.777 2.35E-05arachidonate 5-lipoxygenase NEU4 -3.361 8.31E-40 neuraminidase 4(sialidase) GAL3ST1 -2.681 4.01E-12 galactose-3-O-sulfotransferase 1PLPPR1 -2.652 1.16E-09 phospholipid phosphatase related 1 PLPPRS -1.5731.69E-09 phospholipid phosphatase related 5 PTPRT 4.410 1.69E-17 proteintyrosne phosphatase, receptor type T PPPIR16B -2.665 1.29E-09 proteinphosphatase 1 regulatory subunit 16B PPAPDC1A -2.357 6.76E-09phospholipid phosphatase 4 DGKG -2.306 9.29E-12 dacylglycerol kinasegamma EPHB1 -1.344 3.89E-17 EPH receptor B1 PNCK -1.226 4.25E-08pregnancy up-regulated nonubiquitous CaM kinase D SCAM -3.148 1.586-12Down syndrome cell adhesion molecule ASTN2 -2.242 4.70E-21 astrotactin 2OPCML -2.099 7.87E-10 opioid binding protein/cell adhesion molecule-likeBAI1 -2.000 1.31E-12 adhesion G protein-coupled receptor B1 CDH10 -1.6469.33E-07 cadherin 10 CCL2 1.832 1.97E-07 chemokine (C-C motif) ligand 2GPR17 -3.357 1.94E-10 G protein-coupled receptor 17 GPR45 -2.8952.95E-14 G protein-coupled receptor 45 WNT7B -2.626 1.46E-07wingless-type MMTV integration site family member 78 GPR139 -2.5891.32E-08 G protein-coupled receptor 139 OMG -2.561 5.09E-12digodendrocyte myelin glycoprotein CRHR1 -2.382 5.81E-12 corticotropinreleasing hormone receptor 1 DGKG -2.306 9.29E-12 dacylglycerol kinasegamma BAI1 -2.000 1.31E-12 adhesion G protein-coupled receptor B1 GPR123-1.807 1.76E-18 adhesion G protein-coupled receptor A1 (continued on thenew page) Gene ID Log2 FC P Value Entrez Gene Name FGF14 -2.021 5.14E-07fibroblast growth factor 14 FGF12 -1.988 5.47E-09 fibroblast growthfactor 12 CSPG5 -1.285 6.38E-17 chondroitin sulfate proteoglycan 5 CCL21.832 1.97E-07 chemokine (C-C motif) ligand 2 CMTM5 -3.023 3.69E-15CKLF-like MARVEL transmembrane domain containing 5 CCL2 1.832 1.97E-07chemokine (C-C motif) ligand 2 ALOX5 1.777 1.777 2.35E-05 aradidonate5-lipoxygenase ALOX5 BRINP3 -3.433 4.70E-22 bone morphogeneticprotein/retinoic acid inducible neural-specific 3 DSCAM -3.148 1.58E-12Down syndrome cell adhesion molecule KCND 2 -3.145 5.67E-11 potassiumchannel, voltage gated Shal related subfamily D. member 2 NXPH1 CHRNA4-2.892 3.11E-15 neurexophilin 1 CHRNA4 -2.755 4.45E-14 cholinergicreceptor, nicotinic alpha 4 ARHGAP36 CNTN 1 -2.686 -2.675 1.21E-055.65E-15 Rho GTPase activating protein 36 contactin 1 NETO1 -2.6332.52E-12 neuropilin and tolloid like 1 PLLP -2.581 1.21E-24 plasmolipinPCDH15 -2.530 1.42E-10 protocadherin-related 15 GRIA4 -2.519 1.84E-09glutamate receptor, ionotropic, AMPA 4 BCAN -2.473 7.35E-32 brevicanGABRA3 -2.450 1.08E-12 gamma-aminobutyric and (GABA) A receptor, alpha 3CRHR1 -2.382 5.81E-12 corticotropin releasing hormone receptor 1 SHISA7-2.302 1.43E-14 shisa family member 7 SLC8A3 -2.224 2.00E-12 solutecarrier family 8 (sodium/calaum exchanger), member 3 MPZ -2.105 7.66E-13myelin protein zero GRID2 -2.055 3.65E-06 glutamate receptor,ionotropic, delta 2 RPH3A -2.014 1.01E-06 rabphilin 3A VWC2 -2.0039.67E-13 von Willebrand factor C domain containing 2 CTTNBP2 -1.9047.21E-08 cortactin bioding protein 2 MT3 -1.795 5.27E-09 metallothionein3 KCNA3 -1.719 3.03E- 12 potassium channel, voltage gated shaker relatedsubfamily A, member 3 BRINP2 -1.625 2.03E-12 bone morphogeneticprotein/retinoic acid inducible neural-specific 2 LGI3 -1.614 4.07E-08leucine-rich repeat LGI family member 3 SLC6A1 -1.596 5.52E-11 solutecarrier family 6 (neurotransmitter transporter), member 1 GRID 1 GRIK4-1.562 -1.547 7.61E-07 2.73E-07 glutamate receptor, ionotropic, delta 1glutamate receptor, ionotropic, kainate 4 KCNK9 -1.460 4.40E-06potassium channel, two pore domain subfamily K, member 9 EPHB1 -1.3443.89E-17 EPH receptor B1 KCND2 -3.145 5.67E-11 potassium channel,voltage gated Shal related subfamily D, member 2 GRIA4 -2.519 1.84E-09glutamate receptor, ionotropic, AMPA 4 GABRA3 -2.450 1.08E- 12gamma-aminobutync acid (GABA) Areceptor, alpha 3 ASIC4 -2.321 4.13E-14acid sensing ion channel subunit family member 4 KCNJ9 -2.187 2.85E-21potassium channel, inwardly rectifying subfamily J, member 9 GRID2-2.055 3.65E-06 glutamate receptor, ionotropic, delta 2 KCNA3 -1.7193.03E-12 potassium channel, voltage gated shaker related subfamily A,member 3 GRID 1 -1.562 7.61E-07 glutamate receptor, ionotropic, delta 1GRIK4 -1.547 2.73E-07 glutamate receptor, ionotropic, kainate 4 KCNK9-1.460 4.40E-06 potassium channel, two pore domain subfamily K, member 9(continued on next page) AZGP1 -3.323 1.66E-08 alpha-2-glycoprotein 1,zinc-binding PLLP -2.581 1.21E-24 plasmolipin SNX22 -2.297 3.53E-20sorting nexin 22 SLC8A3 -2.224 2.00E-12 solute carrier family 8(sodium/calcium exchanger), member 3 RPH3A -2.014 1.01E-06 rabphilin 3ASLC6A1 -1.596 5.52E-11 solute carrier family 6 (neurotransmittertransporter), member 1 CCL2 1.832 1.97E-07 chemokine (C-C motif) ligand2 COL20A1 -3.476 7.33E-14 collagen, type XX, alpha 1 BRINP3 -3.4334.70E-22 bone morphogenetic protein/retinoic acid inducibleneural-specific 3 WNT7B -2.626 1.46E-07 wingless-type MMTV integrationsite family member 7B BCAN -2.473 7.35E-32 brevican FGF14 -2.0215.14E-07 fibroblast growth factor 14 VWC2 -2.003 9.67E-13 von Willebrandfactor C domain containing 2 FGF12 -1.988 5.47E-09 fibroblast growthfactor 12 LUZP2 -1.948 7.22E-06 leucine zipper protein 2 ELFN2 -1.9431.47E-06 extracellular Leu-rich repeat and fibronectin type III domaincontaining 2 KY -1.873 2.41E-13 kyphoscoliosis peptidase BRINP2 -1.6252.03E-12 bone morphogenetic protein/rebnoic acid inducibleneural-specific 2 LGI3 -1.614 4.07E-08 leucine-rich repeat LGI familymember 3 CSPGS -1.285 6.38E-17 chondroitin sulfate proteoglycan 5TMEM257 -4.018 5.58E-23 transmembrane protein 257 CMTM5 -3.023 3.69E-15CKLF-like MARVEL transmembrane domain containing 5 CHRNA4 -2.7554.45E-14 cholinergic receptor, nicotinic alpha 4 LINGO1 -2.139 3.56E-14leucine-rich repeat and 1g domain containing 1 TME229B -1.187 7.76E-06transmembrane protein 229B CCL2 1.832 1.97E-07 chemokine (C-C motif)ligand 2 BTN3A2 1.355 8.10E-20 butyrophilin subfamily 3 member A2 BEX5-3.885 2.55E-21 brain expressed X-linked 5 ARHGAP36 -2.686 1.21E-05 RhoGTPase activating protein 36 WNT7B -2.626 1.46E-07 wingless-type MMTVintegration site family member 7B GRIA4 -2.519 1.84E-09 glutamatereceptor, ionotropic, AMPA 4 SNX22 -2.297 3.53E-20 sorting nexin 22LHFPL3 -2.250 1.71E-07 lipoma HMGIC fusion partner-like 3 RPH3A -2.0141.01E-06 rabphilin 3A CTTNBP2 -1.904 7.21E-08 cortactin binding protein2 SHC3 -1.830 7.88E-19 SHC (Src homology 2 domain containing)transforming protein 3 MT3 -1.795 5.27E-09 metallothionein 3 LGI3 -1.6144.07E-08 leucine-rich repeat LGI family member 3 KCNK9 -1.460 4.40E-06potassium channel, two pore domain subfamily K, member 9 CSPGS -1.2856.38E-17 chondroitin sulfate proteoglycan 5 (continued on next page)RPS12P11 1.647 9.02E-30 not available LOC101927206 -3.592 2.74E-63 notavailable LOC100505797 -3.266 3.10E-27 myosin heavy chain IB-likeC10orf85 -3.081 3.52E-10 long intergenic non-protein coding RNA 1561CXXC11 -2.814 7.70E-19 receptor (chemosensory) transporter protein 5(putative) LOCZ84395 -2.792 1.20E-10 uncharacterized LOC284395RP11-547I7.2 -2.779 4.81E-34 not available AC009227.2 -2.758 3.05E-13not available LOC101928430 -2.691 5.56E-10 not available FLJ38379 -2.3891.25E-15 not available LOC102723927 -2.363 7.74E-21 uncharacterizedLOC102723927 RP11-1G5598.3 -2.336 1.02E-16 not available AC131097.3-2.180 5.80E-16 not available C1orf94 -2.077 7.03E-11 chromosome 1 openreading frame 94 HMGA1P7 -2.057 4.49E-09 high mobility group AT-hook 1pseudogene 7 LOC101929249 -2.003 1.52E-06 uncharacterized LOC101929249LOC101927646 -2.001 1.22E-10 uncharacterized LOC101927646 AC010890.1-1.937 3.53E-10 not available C2orf80 -1.924 6.74E-18 chromosome 2 openreading frame 80 MAPT-AS1 -1.844 5.40E-07 MAPT antisense RNA 1LOC100128127 -1.814 1.43E-09 not available RP11-1134114.8 -1.7835.72E-22 not available 5LC6A1-4S1 -1.706 1.31E-09 SLC6A1 antisense RNA 1KIAA1244 -1.643 1.63E-10 ARFGEF family member 3 F AM222A-AS1 -1.2971.10E-11 FAM222A antisense RNA 1 Table 2. Significantly dysregulatedgenes in SCZ relative to control GPCs. These tables list shared genesdifferentially expressed by hiPSC GPCs derived from 4 schizophrenicpatients, relative to the pooled gene expression pattern of hGPCsderived from 3 control-derived iPSCs (log2 fold change > 1.0, FDR 5%,116 genes total, red, upregulated in SCZ vs CTRL; green, downregulatedin SCZ GPCs; color intensity proportionate to differentialdysregulation). The fold- changes (FC) and FDR-adjusted p values shownhere were derived from the comparison of the pooledschizophrenia-derived GPC cell lines to the pooled control-derived GPClines. The dysregulated genes were grouped into functional setsaccording to their cellular roles and localizations.

These expression data suggest that the diminished myelination of SCZhGPC-transplanted shiverer brains reflected aberrant oligodendrocyticdifferentiation from the engrafted SCZ hGPCs. Similarly, since hGPCsgive rise to astrocytes as well as oligodendrocytes, the RNA expressiondata suggest an analogous impediment to astrocytic differentiation. Thefunctional consequences of the latter are especially profound, given thecritical role for astrocytes in synaptic development and function;indeed, the relative suppression of astrocytic differentiation by SCZhGPCs suggests a glial contribution to the impaired synaptic functionnoted in schizophrenia. In that regard, further functional analysis ofSCZ-associated dysregulated hGPC genes identified channel and receptoractivity, as well as synaptic transmission, as the most differentiallyaffected functions besides glial differentiation (FIGS. 6D-6E). Thesedisease-linked channel and synapse-associated genes were largelydown-regulated in the SCZ hGPCs, and included a number of potassiumchannel genes (FIG. 6D), including KCND2, KCNJ9, KCNK9 and KCNA3, aswell as a number of transcripts associated with synaptic development andfunction (FIG. 6E and Table 2). The latter included NXPH1, NLGN3, andLINGO1, among others (Table 3), synaptic genes whose dysregulation hasbeen previously linked to both SCZ and the autism spectrum disorders(Sudhof, T. C., “Neuroligins and Neurexins Link Synaptic Function toCognitive Disease,” Nature 455:903-911 (2008); Andrews et al., “A DecadeFrom Discovery to Therapy: Lingo-1, the Dark Horse in Neurological andPsychiatric Disorders,” Neurosci Biobehav Rev 56:97-114 (2015);Fernandez-Enright et al., “Novel Implications of Lingo-1 and itsSignaling Partners in Schizophrenia,” Translational psychiatry 4:e348(2014); Mackowiak et al., “Neuroligins, Synapse Balance andNeuropsychiatric Disorders,” Pharmacol Rep 66:830-835 (2014); Salyakinaet al., “Copy Number Variants in Extended Autism Spectrum DisorderFamilies Reveal Candidates Potentially Involved in Autism Risk,” PloSone 6:e26049 (2011), which are hereby incorporated by reference in theirentirety).

TABLE 3 CZ.08 vs. Pooled CTR SCZ.29 vs. Pooled CTR SCZ.51 vs. Pooled CTRSCZ.164 vs. Pooled CTR SCZ.08+29+S1+164 vs. Pooled CTR Gene ID Log2 FC PValue Log2 FC P Value Log2 FC P Value Log2 FC P Value Log2 FC P ValueDSCAML1 -1.971 8.83E-07 -2.968 1.31E-04 -1.089 8.32E-05 NS NS -0.9825.34E-08 LINGO1 -2.320 3.29E-04 -2.885 2.26E-04 -1.452 3.36E-06 -2.5239.19E-12 -2.139 3.56E-14 NLGN1 -1.249 3.37E-04 -1.014 4.32E-02 NS NS-0.545 3.88E-02 -0.625 3.15E-04 NLGN2 NS NS -0.767 1.74E-02 -0.3843.01E-03 -0.408 1.47E-02 -0.452 3.37E-06 NLGN3 -0.563 3.98E-02 -1.6693.07E-03 -1.011 1.63E-09 -0.958 3.67E-05 -1.143 1.61E-19 NRP1 -1.3621.15E-03 1.409 5.00E-04 NS NS NS NS NS NS NRP2 NS NS 1.548 2.07E-03 NSNS -0.733 3.65E-02 NS NS NRXN1 -3.259 5.93E-06 -2.984 5.55E-03 -1.1761.35E-02 NS NS -1.161 2.04E-04 NRXN2 NS NS -2.179 9.04E-06 -1.2522.71E-11 -0.720 3.33E-03 -1.102 6.82E-17 NRXN3 1.874 1.60E-04 NS NS NSNS 1.198 1.20E-02 0.909 6.89E-04 NTNG2 -0.874 7.99E-03 -1.814 3.51E-04NS NS NS NS NS NS NXPE3 NS NS NS NS NS NS 0.410 1.74E-02 0.248 2.79E-02NXPH1 -3.019 3.98E-07 -3.666 1.84E-03 -1.338 1.06E-02 -2.317 2.46E-09-2.892 3.11E-15 NXPH2 -2.288 5.62E-04 NS NS NS NS NS NS NS NS NXPH3 NSNS -2.225 1.49E-03 -1.084 7.92E-03 NS NS -0.675 2.66E-02 NXPH4 -2.1865.96E-03 4.095 4.08E-14 NS NS -1.560 1.09E-02 NS NS PTPRZ1 NS NS -2.9672.60E-05 -0.792 4.13E-03 -0.870 5.38E-03 -1.296 2.41E-11 RGS4 -1.9826.80E-05 2.184 3.97E-04 1.105 7.04E-04 -1.419 2.64E-03 NS NS SLITRK2-6.812 1.35E-04 NS NS -7.307 7.51E-25 -9.321 5.54E-06 -6.138 5.52E-14SLITRK3 -2.958 2.68E-03 NS NS -1.698 6.37E-04 -3.490 1.61E-11 -2.5026.76E-07 SLITRK4 -4.157 7.12E-05 NS NS -3.713 5.02E-05 -2.678 1.20E-02-2.457 5.86E-05 SLITRK5 -2.047 3.87E-07 NS NS -1.184 1.54E-06 -1.7349.31E-08 -1.152 1.05E-08 SPARCL1 -2.314 6.14E-06 NS NS NS NS -0.8433.87E-02 NS NS TNR -3.082 8.83E-06 -5.108 1.89E-07 -2.227 8.81E-13 NS NS-2.137 5.56E-12 Table 3. Genomic analysis of SCZ-derived hGPCs from 4different patients revealed the significant and shared down-regulationin these cells of a number of synaptic genes, including neuroligin-3,neuroexophilin-1, LINGO1 and DSCAML1, relative to their normal controls(red, upregulated in SCZ vs CTRL; green, downregulated in SCZ GPCs;color intensity proportionate to differential dysregulation). Othersynapse-associated genes, such as the SLITRKs 2-5, were significantlyand sharply downregulated in GPCs derived from 3 of the 4 patients(lines 8, 51 and 164). Lines 08, 29, 51 and 164: schizophrenia-derived,different patients; pooled controls, 3 lines, each from a differentpatient. Individual SCZ line data shown as well as pooled SCZ data, tohighlight both commonalities and distinctions between SCZ GPCs derivedfrom different patients. Log2FC: log₂ fold-change in expression. NS: notsignificant.

Whereas the expression of these latter genes was suppressed in hGPCsderived from all 4 SCZ patients, other synapse-associated genes, such asNRXN1, NLGN1, DSCAML1, and the SLITRKs 2-5, were sharply down-regulatedin hGPCs derived from 3 of the 4 patients, but not in the fourth (Table3). Yet other synapse-associated transcripts, like NXPH3 and NTRNG2,were similarly down-regulated in some patients, but not others. TaqManlow density arrays were used for quantitative real-time PCR validationof these and other dysregulated transcripts of interest, and validatedthe significant differential down-regulation of these differentiationand synaptic function-associated genes (Table 4 and FIG. 8 ).

Together, these data suggest the importance of glial-associated synapticgene expression in schizophrenia, while emphasizing the heterogeneity ofpathways that might be mechanistically complicit in its dysregulation.These data also highlight the point that while the neuronal localizationof these synaptic proteins has long been recognized, their synthesis byglia and synaptic contributions thereof have not been specificallydiscussed, although cell type-specific transcriptional databases havenoted significant glial expression of these genes (Zhang et al., “AnRNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, andVascular Cells of the Cerebral Cortex,” The Journal of Neuroscience :The Official Journal of the Society for Neuroscience 34:11929-11947(2014), which is hereby incorporated by reference in its entirety).Since NRXN1, a synapse-associated transcript closely linked toschizophrenia (Sudhof, T. C., “Neuroligins and Neurexins Link SynapticFunction to Cognitive Disease,” Nature 455:903-911 (2008), which ishereby incorporated by reference in its entirety), was one of the moststrongly and consistently down-regulated glial genes across thepatients, the down-regulation of its expression by SCZ glia wasverified, by immunoblotting CD140a-sorted, neuron-free isolates of SCZand control hGPCs. Western blots revealed that neurexin-1 was indeedabundantly expressed by human GPCs, and that neurexin-1 protein levelswere sharply lower in otherwise matched SCZ hGPCs (FIG. 9 ).

TABLE 4 Gene Symbol qPCR Ratio (min-max; P Value) Transcriptionregulators LINGO1*** 0.105 (0.064 - 0.174; P = 2.20E-03) MYRF 0.263(0.092 - 0.754; P = 1.11E-01) NKX2-2** 0.253 (0.115 - 0.556; P =2.17E-02) OLIG1*** 0.170 (0.076 - 0.381; P = 4.83E-03) OLIG2*** 0.119(0.053 - 0.268; P = 1.09E-03) SOX10*** 0.049 (0.013 - 0.178; P =1.09E-03) SOX9* 0.692 (0.524 - 0.914; P = 7.76E-02) TCF7L2* 0.639(0.451 - 0.906; P = 7.70E-02) ZNF488** 0.130 (0.042 - 0.397; P =2.12E-02) Myelination-associated CNTN1*** 0.076 (0.046 - 0.127; P =9.83E-04) FA2H*** 0.040 (0.010 - 0.171; P = 1.34E-03) GPR17** 0.094(0.019 - 0.467; P = 2.18E-02) MPZ 0.482 (0.211 - 1.103; P = 4.99E-01)OMG*** 0.154 (0.082 - 0.287; P = 1.09E-03) SIRT2*** 0.401 (0.307 -0.524; P = 4.54E-03) UGT8*** 0.045 (0.012 - 0.160; P = 1.09E-03)Synaptic junction-associated ATP2B2*** 0.209 (0.113 - 0.385; P =2.49E-03) BCAN*** 0.191 (0.113 - 0.323; P = 2.49E-03) CD44** 2.397(1.435 - 4.007; P = 3.19E-02) CRHR1** 0.183 (0.058 - 0.583; P =2.24E-02) DSCAML1*** 0.302 (0.190 - 0.480; P = 2.49E-03) LGI1 0.821(0.228 - 2.957; P = 8.10E-01) NETO1*** 0.095 (0.042 - 0.214; P =9.83E-04) NRXN1** 0.252 (0.124 - 0.510; P = 2.12E-02) NTNG1** 0.348(0.149 - 0.817; P = 4.69E-02) NTNG2 0.569 (0.223 - 1.451; P = 3.66E-01)NXPH1*** 0.085 (0.034 - 0.216; P = 1.30E-03) RPH3A*** 0.205 (0.130 -0.324; P = 3.24E-03) SLC6A1*** 0.188 (0.094 - 0.378; P = 4.44E-03)SLITRK2** 0.010 (0.003 - 0.033; P = 3.19E-02) SLITRK3*** 0.074 (0.025 -0.220; P = 1.33E-03) SLITRK4* 0.101 (0.030 - 0.335; P = 5.73E-02)SPARCL1* 0.507 (0.275 - 0.936; P = 9.14E-02) TNR*** 0.102 (0.045 -0.228; P = 9.83E-04) Ion channels KCNA3** 0.270 (0.166 - 0.439; P =1.30E-02) KCND2*** 0.070 (0.025 - 0.196; P = 1.09E-03) KCNH8 0.534(0.261 - 1.093; P = 2.56E-01) KCNJ9** 0.257 (0.151 - 0.439; P =2.24E-02) KCNK9** 0.159 (0.061 - 0.411; P = 1.28E-02) Actin ACTB 1.365(1.016 - 1.833; P = 1.27E-01) Astrocyte-specific marker GFAP 1.479(0.819 - 2.672; P = 4.25E-01) Enzyme ALDH1L1 0.596 (0.164 - 2.170; P =5.67E-01) Growth factor FGF14*** 0.085 (0.034 - 0.213; P = 1.33E-03) RNAbinding protein ELAVL4 0.585 (0.374 - 0.915; P = 1.61E-01) Wnt signalingWNT7B* 0.149 (0.042 - 0.527; P = 5.43E-02) Table 4. Expression ofselected genes identified by RNA-seq analysis as dysregulated inSCZ-derived GPCs was assessed by TaqMan Low Density Array (TLDA)RT-qPCR, and compared to that of control GPCs. Expression data werenormalized to GAPDH endogenous control. Mean expression ratioscalculated from 4 pooled SCZ GPC lines (n = 19) against 3 pooled controlGPC lines (n = 10) are shown. The difference of expression in SCZ andcontrol GPCs was assessed by paired t-test followed by multiple testingcorrection by Benjamini-Hochberg (BH) procedure. BH-corrected P valuesare shown (*** = P < 0.01, ** = P < 0.05, * = P < 0.1). 48 genes wereassessed. 45 genes are shown, excluding the endogenous control and genesthat had high proportion of undetermined and unreliable reactions, LRFN1and NEUROD6. The vast majority of genes were confirmed as dysregulatedin SCZ-derived GPCs which reliably exhibited the significantdifferential down-regulation of differentiation, potassium channel andsynapse function-associated genes. Analysis of TLDA data was performedin ExpressionSuite Software version 1.1 supplied by Applied Biosciences.

Example 5 — SCZ Glial Chimerization Yielded Disease-Specific BehavioralPhenotype

It was next asked whether the alterations in glial distribution anddifferentiation observed in mice engrafted with SCZ hGPCs might alterthe behavioral phenotype of the host mice. In particular, it waspostulated that the aberrant infiltration of hGPCs and their derivedastroglia into the developing cortex might influence informationprocessing within the cortex once mature. As noted, past studies havereported both the influence of astrocytic networks on synaptic efficacyand plasticity, and the differential competence of hominid glia in thisrespect (Oberheim et al., “Uniquely Hominid Features of Adult HumanAstrocytes,” J. Neurosci. 29:3276-3287 (2009); Han et al., “ForebrainEngraftment by Human Glial Progenitor Cells Enhances Synaptic Plasticityand Learning in Adult Mice,” Cell Stem Cell 12:342-353 (2013), which arehereby incorporated by reference in their entirety). Human glialchimeric mice manifest a lower threshold for hippocampal long-termpotentiation (LTP) and learn more rapidly, with superior performance ina variety of learning tasks, which include auditory fear conditioning,novel object and place recognition, and Barnes maze navigation. In eachof these tests - but not in any test of social interactivity or primaryperception - human glial chimeras acquire new causal associations morequickly than do allografted or untransplanted controls (Han et al.,“Forebrain Engraftment by Human Glial Progenitor Cells Enhances SynapticPlasticity and Learning in Adult Mice,” Cell Stem Cell 12:342-353(2013), which is hereby incorporated by reference in its entirety).Thus, engrafted human GPCs and their daughter glia can integrate into,and substantially modify, developing neural networks (Franklin et al.,“Do Your Glial Cells Make You Clever?,” Cell Stem Cell 12:265-266(2013), which is hereby incorporated by reference in its entirety). Onthat basis, it was postulated that the disruption in normal glialdevelopment noted in the SCZ glial chimeras might yielddisease-associated changes in learning and behavior. To address thisquestion, the behavioral phenotypes of immunodeficient but otherwisewild-type mice neonatally engrafted with SCZ GPCs were assessed,relative to matched hosts engrafted with control-derived GPCs. For theseexperiments, normally-myelinated hosts were used rather than shiverermice, so as to produce mice chimeric only for human GPCs and astrocytes,and not for oligodendroglia, thus isolating any observed behavioraleffects to SCZ hGPCs and astrocytes.

It was first asked whether schizophrenic derivation of engrafted gliaaffected prepulse inhibition (PPI), a behavioral hallmark of bothclinical schizophrenics and animal models thereof (Ewing et al.,“Evidence for Impaired Sound Intensity Processing During PrepulseInhibition of the Startle Response in a Rodent Developmental DisruptionModel of Schizophrenia,” Journal of Psychiatric Research (2013), whichis hereby incorporated by reference in its entirety). PPI reflects thecoordination of sensorimotor gating in the CNS, and its diminution maypredict aspects of schizophrenic phenotype (Ivleva et al., “SmoothPursuit Eye Movement, Prepulse Inhibition, and Auditory Paired StimuliProcessing Endophenotypes Across the schizophrenia-Bipolar DisorderDimension,” Schizophrenia Bulletin (2013); Kohl et al., “PrepulseInhibition in Psychiatric Disorders--Apart from Schizophrenia,” Journalof Psychiatric Research 47:445-452 (2013), which is hereby incorporatedby reference in its entirety). It was found that when assessed at 6months of age - the latest time-point at which the C57Bl/6 backgroundstrain of the rag1^(-/) ⁻ mice can be reliably assessed, since thesemice suffer premature auditory loss which might otherwise diminishauditory PPI - that mice engrafted with SCZ hGPCs exhibitedsignificantly diminished auditory prepulse inhibition (FIG. 10A), anddid so at all volumes of pre-pulse. Given the strong effect of SCZ glialchimerization on PPI, it was next asked if SCZ glial chimerization mightbe associated with changes in behavior on cognitive and socializationtests. To that end, SCZ and control chimeras were compared on a batteryof behavioral tests that included: 1) the elevated plus maze, a measureof anxiety (Walf et al., “The Use of the Elevated Plus Maze as an Assayof Anxiety-Related Behavior in Rodents,” Nat Protoc 2:322-328 (2007),which is hereby incorporated by reference in its entirety); 2) the3-chamber social challenge (Yang et al., “Automated Three-ChamberedSocial Approach Task for Mice,” Curr Protoc Neurosci, Chapter 8, Unit 8,26 (2011), which is hereby incorporated by reference in its entirety);3) novel object recognition, a focused measure of executive memory(Bevins et al., “Object Recognition in Rats and Mice: A One-TrialNon-Matching-to-Sample Learning Task to Study ‘Recognition Memory’,”NatProtoc 1:1306-1311 (2006), which is hereby incorporated by referencein its entirety), and 4) the preference for sucrose water, a test foranhedonia (Barnes et al., “Anhedonia, Avolition, and AnticipatoryDeficits: Assessments in Animals with Relevance to the Negative Symptomsof Schizophrenia,” Eur Neuropsychopharmacol 24:744-758 (2014); Willneret al., “Reduction of Sucrose Preference by Chronic Unpredictable MildStress, and its Restoration by a Tricyclic Antidepressant,”Psychopharmacology (Berl) 93:358-364 (1987), which are herebyincorporated by reference in their entirety). In each, mice chimerizedwith one of 3 SCZ or 3 control patient-derived lines were compared; eachline was derived from a different patient. Between 6-12 recipient micewere engrafted and tested per cell line, or 17-36 mice per group foreach behavioral comparison, with a typically equal balance of male andfemale recipients. These animals were tested beginning between 30-36weeks of age, and testing typically lasted 3 weeks. Over the tested agerange, the SCZ GPC chimeric mice exhibited a number of significantdifferences in behavior relative to their control hGPC-engraftedcounterparts. Normal control-engrafted mice are significantly morelikely to explore the open arms (horizontal segments), whereas SCZ micespent most of their time in the closed maze arms (vertical segments),consistent with greater anxiety (p=0.036, 2-tailed t test). The SCZ hGPCmice exhibited greater avoidance of the open arms in the elevated plusmaze than did their normal hGPC-engrafted controls (n=36 mice/group,each including 12 mice engrafted with hGPCs from each of 3 patients;p=0.036, 2-tailed t test), suggesting that the SCZ hGPC mice were proneto higher anxiety when challenged (FIG. 10B). In addition, the SCZ hGPCmice showed less preference for sucrose water), consistent with relativeanhedonia (FIG. 10C), less interest in stranger mice in the 3-chambersocial test (FIG. 10D), and relatively poor novel object recognition(FIG. 10E), reflecting relative impairment in executive memory.

As an additional metric of SCZ-associated behavior, sleep and diurnalactivity patterns of human SCZ and CTRL glial chimeras were thenassessed, directly comparing mice engrafted with either SCZ (line 52) ormatched control (line 22) hGPCs. It was found that mice engrafted withSCZ GPCs were significantly more active than control mice engrafted withnormal hGPCs. As measured by meters moved per hour, over the course of a72-hour video-recording (Noldus Ethovision), the SCZ hGPC chimeric micemoved significantly more than their normal hGPC-engrafted controls(2-way ANOVA, F=48.35; p<0.0001) (FIG. 10F). Interestingly, while theSCZ-associated increment in activity largely occurred during night-timeperiods of wakefulness, the SCZ mice also manifested disrupted sleeppatterns, as measured by the duration of bouts of inactivity, asurrogate for EEG-validated sleep (Pack et al., “Novel Method forHigh-Throughput Phenotyping of Sleep in Mice,” Physiol. Genomics28:232-238 (2007) ; McShane et al., “Characterization of the BoutDurations of Sleep and Wakefulness,” J. Neurosci. Methods 193:321-333(2010), which are hereby incorporated by reference in their entirety)(FIG. 10G). Within the half-hour following the phase transition fromdark to light (when mice normally sleep), the CTRL mice had morecontinuous, uninterrupted patterns of sleep, with an average sleep boutof 511.5 ± 36.4 seconds (8.53 minutes), whereas SCZ mice were asleep for306.2 ± 43.7 seconds, or 5.1 minutes per bout (p<0.01 by 2-way ANOVA,with Boneferroni post hoc t tests). The shorter average periods ofinactivity manifested by SCZ hGPC mice during the normal daytimetransition to sleep suggests that SCZ hGPC chimerization disruptednormal daytime sleep patterns, while increasing night-time activity.Together, these results suggest that SCZ glial chimerization wassufficient to yield heightened anxiety and fear in engrafted recipients,as well as disease-associated deficits in socialization, cognition, andsleep patterning, all features associated with human schizophrenia.

Discussion of Examples 1-7

These data suggest a significant contribution of cell-autonomous glialpathology to the genesis and development of juvenile-onsetschizophrenia. In these human glial chimeric mice, schizophrenia-derivediPSC hGPCs exhibited aberrant migration with deficient engraftment inthe central white matter, relative to age and gender-matched controliPSC hGPCs. Although a fraction of those SCZ hGPCs that did remainwithin the white matter differentiated as normal myelinogenicoligodendroglia, the premature cortical influx and hence lower densityof donor-derived cells in the white matter of SCZ hGPC-engrafted miceresulted in the latter’s overt hypomyelination, relative to miceengrafted with control GPCs. Thus, SCZ hGPCs appeared to traverse ratherthan home in to the nascent white matter, resulting in sparse hGPCcolonization and hence deficient forebrain myelination. The aberrantdispersal pattern of SCZ hGPCs suggests that SCZ GPCs may not recognizedevelopmental stop signals that permit progenitors to dwell and expandwithin the presumptive white matter before colonizing the corticalmantle, and may instead be biased towards rapid entry into the corticalgray matter. These observations in human SCZ glial chimeric mice areespecially intriguing given the well-described hypomyelination ofschizophrenic patients (Voineskos et al., “Oligodendrocyte Genes, WhiteMatter Tract Integrity, and Cognition in Schizophrenia,” Cereb Cortex23:2044-2057 (2013); Najjar et al., “Neuroinflammation and White MatterPathology in Schizophrenia: Systematic Review,” Schizophrenia Research161:102-112 (2015); Davis et al., “White Matter Changes inSchizophrenia: Evidence for Myelin-Related Dysfunction,” Archives ofGeneral Psychiatry 60:443-456 (2003); Sigmundsson et al., “StructuralAbnormalities in Frontal, Temporal, and Limbic Regions andInterconnecting White Matter Tracts in Schizophrenic Patients withProminent Negative Symptoms,” Am J Psychiatry 158:234-243 (2001), whichare hereby incorporated by reference in their entirety), particularly soin early onset disease (Gogtay et al., “Three-Dimensional Brain GrowthAbnormalities in Childhood-Onset Schizophrenia Visualized by UsingTensor-Based Morphometry,” Proceedings of the National Academy ofSciences of the United States of America 105:15979-15984 (2008);Samartzis et al., “White Matter Alterations in Early Stages ofSchizophrenia: A Systematic Review of Diffusion Tensor Imaging Studies,”J Neuroimaging 24:101-110 (2014); Gogtay et al., “Childhood-OnsetSchizophrenia: Insights From Neuroimaging Studies,” Journal of theAmerican Academy of Child and Adolescent Psychiatry 47:1120-1124 (2008),which are hereby incorporated by reference in their entirety).

These anatomic observations were especially intriguing in light of thedifferential gene expression pattern of the SCZ hGPCs, which revealedthat the cells were deficient not only in early glialdifferentiation-associated transcripts, but also in genes that encodefor synaptic proteins typically associated with transducingactivity-dependent signals (Sudhof, T. C., “Neuroligins and NeurexinsLink Synaptic Function to Cognitive Disease,” Nature 455:903-911 (2008),which is hereby incorporated by reference in its entirety). Together,these anatomic and transcriptional data suggest that SCZ hiPSC-derivedGPCs might be subject to impaired phenotypic differentiation, that mightresult in their neglect of the local neuronal signals that typicallyregulate the expansion and maturation of GPCs (Barres et al.,“Proliferation of Oligodendrocyte Precursor Cells Depends on ElectricalActivity in Axons,” Nature 361:258-260 (1993), which is herebyincorporated by reference in its entirety); this might account for theirrapid transit through the white matter into the overlying cortex, andhence the diminished callosal GPC density and hypomyelination of SCZchimeric shiverer mice (FIG. 3 ). Thus, the myelination defect in SCZhGPC chimeras appeared due to both deficient oligodendrocyticdifferentiation and the relative dearth of SCZ hGPCs remaining withinthe white matter. Moreover, astrocytic differentiation from SCZ hGPCswas also impaired, and may have contributed further to hypomyelinationin the SCZ glial chimeras, given the metabolic dependence of matureoligodendrocytes upon local astrocytes (Amaral et al., “MetabolicAspects of Neuron-Oligodendrocyte-Astrocyte Interactions,” FrontEndocrinol (Lausanne) 4:54 (2013); John, G. R., “Investigation ofAstrocyte - Oligodendrocyte Interactions in Human Cultures,” Methods MolBiol 814:401-414 (2012), which is hereby incorporated by reference inits entirety).

Importantly, the defective astrocytic maturation of SCZ hGPCs might alsohave profound effects on developmental synaptogenesis and circuitformation, as well as on myelinogenesis. Neural connectivity andsynaptic development are both intimately dependent upon astrocyticguidance (Clarke et al., “Glia Keep Synapse Distribution Under Wraps,”Cell 154:267-268 (2013); Ullian et al., “Control of Synapse Number byGlia,” Science 291:657-661 (2001), which is hereby incorporated byreference in its entirety), and hence upon the appropriate timing ofastrocytic appearance and maturation. As a result, any disruption inastrocytic maturation by SCZ hGPCs, as observed in each of the SCZ linesstudied, might be expected to significantly confound the constructionand functional architecture of those neural networks in which SCZ hGPCsare resident. Moreover, glial progenitors themselves may havesignificant interactions with local neurons (Sakry et al.,“Oligodendrocyte Precursor Cells Modulate the Neuronal Network byActivity-Dependent Ectodomain Cleavage of Glial NG2,” PLoS Biol12:e1001993 (2014), which is hereby incorporated by reference in itsentirety), such that their dysfunction might disrupt local neuronalresponse thresholds and circuit formation.

Besides the anatomic observation of deficient astrocytic maturation inSCZ hGPC chimeras, the genomic analysis of SCZ-derived hGPCs revealedthe significant down-regulation in hGPCs derived from all 4 SCZ patientsof a number of synaptic genes, including neuroligin-3, neuroexophilin-1,and LINGO1 relative to their normal controls (Tables 3 and Table 4; FIG.8 ). Other synapse-associated genes, such as neurexin-1 and DSCAML1 weresignificantly and sharply down-regulated in GPCs derived from 3 patients(lines 8, 29, and 51) but not in the fourth (line 164). Similarly,SLITRKs 2-5 were significantly and sharply down-regulated in GPCsderived from 3 patients (lines 8, 51, and 164), but not in a fourth(line 29), which was instead associated with sharp down-regulation ofLINGO1, DSCAML1, and several neurexins and neuroexophilins; these datasuggesting the heterogeneity of transcriptional dysfunction that maylead to a final common pathway of glial-involved synaptic dysfunction inSCZ (Tables 2 and 3). These transcripts are critical contributors tosynaptic stabilization and function (Sudhof, T. C., “Neuroligins andNeurexins Link Synaptic Function to Cognitive Disease,” Nature455:903-911 (2008), which is hereby incorporated by reference in itsentirety), but while typically considered neuronal, may be producedsignificantly by glial cells as well (Zhang et al., “An RNA-SequencingTranscriptome and Splicing Database of Glia, Neurons, and Vascular Cellsof the Cerebral Cortex,” J. Neurosci. 34:11929-11947 (2014), which ishereby incorporated by reference in its entirety). The relativedown-regulation of these genes by SCZ hGPCs may reflect the suppressionof mature glial transcripts in these cells, coincident with theirrelative block in glial differentiation. This in turn may lead to arelative failure of SCZ hGPCs and their derived astrocytes to providethese key proteins to their neuronal partners, as well as a potentialfailure on the part of glial progenitors receiving synaptic inputs torespond to afferent stimulation (De Biase et al., “Excitability andSynaptic Communication Within the Oligodendrocyte Lineage,” J Neurosci30:3600-3611 (2010); Lin et al., “Synaptic Signaling Between GABAergicInterneurons and Oligodendrocyte Precursor Cells in the Hippocampus,”Nat. Neurosci. 7:24-32 (2004), which are hereby incorporated byreference in their entirety). Thus, besides the structural havoc thatmight be expected of a cortical connectome formed without normalastrocytic support, the synaptic structure of the resultant networksmight be expected to be destabilized by poor SCZ glial provision to thesynaptic cleft of key astrocytic proteins required for normal synapticmaintenance and function.

Schizophrenia is genetically heterogeneous, so that anatomic andbehavioral pathology may vary significantly among animals chimerizedwith GPCs derived from different patients. It is thus critical that theresults obtained from chimeras established with control hiPSC GPCs bestable across both distinct lines of donor cells, and among recipientmice. The chimeric brains established from the hGPCs of 3 different SCZpatients were thus compared anatomically to those established from GPCsderived from 3 control patients. None of the controls manifested thewhite matter-avoidant dispersal pattern of the SCZ hGPC chimeras.Similarly, this pattern of SCZ hGPC avoidance of the white matter hadnever been noted in any of several hundred human glial chimerasengrafted in other studies with either fetal tissue-derived (Windrem etal., “Neonatal Chimerization with Human Glial Progenitor Cells Can BothRemyelinate and Rescue the Otherwise Lethally Hypomyelinated ShivererMouse,” Cell Stem Cell 2:553-565 (2008); Windrem et al., “A CompetitiveAdvantage by Neonatally Engrafted Human Glial Progenitors Yields MiceWhose Brains are Chimeric for Human Glia,” J. Neurosci. 34:16153-16161(2014), which are hereby incorporated by reference in their entirety) ornormal iPSC-derived (Wang et al., “Human iPSC-Derived OligodendrocyteProgenitor Cells Can Myelinate and Rescue a Mouse Model of CongenitalHypomyelination,” Cell Stem Cell 12:252-264 (2013), which is herebyincorporated by reference in its entirety) hGPCs.

Besides their clear anatomic phenotype, the SCZ hGPC-chimeric micemanifested robust behavioral phenotypes. They exhibited significantlyattenuated prepulse inhibition relative to control-engrafted mice,relative anhedonia, excessive anxiety, deficient socialization withavoidance of conspecifics, and disrupted patterns of diurnal activityand sleep. These data establish that SCZ glial engraftment may yield anabnormal behavioral phenotype in recipient mice, along behavioral axesthat typify selected aspects of schizophrenic behavioral pathology inhumans. In that regard, while an extensive literature has implicatedGPCs (De Biase et al., “Excitability and Synaptic Communication Withinthe Oligodendrocyte Lineage,” J Neurosci 30:3600-3611 (2010); Bergles etal., “Neuron-Glia Synapses in the Brain,” Brain Res Rev 63:130-137(2010), which are hereby incorporated by reference in their entirety) aswell as astroglia (Kang et al., “Astrocyte-Mediated Potentiation ofInhibitory Synaptic Transmission,” Nature Neuroscience 1:683-692 (1998);Araque et al., “Glutamate-Dependent Astrocyte Modulation of SynapticTransmission Between Cultured Hippocampal Neurons,” European J.Neurosci. 10 (1998), which are hereby incorporated by reference in theirentirety) in the modulation of synaptic plasticity and learning (Han etal., “Forebrain Engraftment by Human Glial Progenitor Cells EnhancesSynaptic Plasticity and Learning in Adult Mice,” Cell Stem Cell12:342-353 (2013), which is hereby incorporated by reference in itsentirety), these data do not implicate one phenotype over the other inthe modulation of behavior by SCZ glial chimerization; the chimeric miceare colonized by both donor-derived human GPCs and their derivedastrocytes. That said, the observations of significant defects in SCZglial maturation shared by hGPCs derived from multiple independentpatients, associated in each with hypomyelination and disruptedastrocytic differentiation, as well as with abnormal behavioralphenotypes in the resultant SCZ GPC chimeras, together suggest a strongcausal contribution of glial pathology to schizophrenia. In addition,these data highlight the potential of disease-specific humanizedchimeras in defining the respective contributions of glial and neuronaldysfunction in the genesis and course of neurological disease.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

What is claimed is: 1-10. (canceled)
 11. A method of treating aneuropsychiatric disorder, said method comprising: selecting a subjecthaving the neuropsychiatric disorder and administering, to the selectedsubject, a potassium (K⁺) channel activator at a dosage effective torestore normal brain interstitial glial K⁺ levels in the selectedsubject and treat the neuropsychiatric disorder, with the proviso thatthe K⁺ channel activator is not a KCNQ channel activator.
 12. The methodof claim 11, wherein the selected subject has dysregulated glial K⁺channel function characterized by defective glial K⁺ conductance,defective glial K⁺ uptake, and/or defective glial K⁺ channel expression.13. The method of claim 11, wherein the K⁺ channel activator increasesthe activity of glial G protein-activated inward rectifier K⁺ channels.14. The method of claim 13, wherein the K⁺ channel activator is selectedfrom the group consisting of flupirtine, nitrous oxide, halothane,17β-estradiol, dithiothreitol, and naringin.
 15. The method of claim 11,wherein the K⁺ channel activator increase the activity of glial K⁺voltage-gated channels.
 16. The method of claim 15, wherein the K⁺channel activator increases the activity of a glial A-type voltage-gatedK⁺ channels.
 17. The method of claim 16, wherein the K⁺ channelactivator isN-[3,5-Bis(trifluoromethyl)phenyl]-N′-[2,4-dibromo-6-(2H-tetrazol-5-yl)phenyl]urea(NS5806).
 18. The method of claim 15, wherein the K⁺ channel activatorincreases the activity of glial delayed rectifier K⁺ channels.
 19. Themethod of claim 11, wherein the K⁺ channel activator increase theactivity of glial tandem pore domain K⁺ channels, including thepotassium leak channels encoded by KCNK1 through KCNK18 inclusive. 20.The method of claim 19, wherein the K⁺ channel activator is selectedfrom the group consisting of halothane, isoflurane, 2-haolgenatedethanols, halogenated methanes, sevoflurane, and desflurane.
 21. Themethod of claim 19, wherein said administering is carried out byinhalation, subcutaneous, intramuscular or intravenous administration.22. The method according to claim 11, wherein the neuropsychiatricdisorder is selected from the group consisting of schizophrenia, autismspectrum disorder, and bipolar disorder.
 23. The method according toclaim 11, wherein the neuropsychiatric disorder is schizophrenia. 24.The method of claim 11, wherein the subject is human.